Reaction products of sodium glucoheptonate with manganese sulfate and sodium hydroxide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms, other

Remarks:

non-specific esterase activity, proliferation rate inhibition

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: raw data not given. However, the available data indicates that the test was well performed and meets general scientific requirements.

Qualifier:

no guideline available

Principles of method if other than guideline:

FDA Method:
T. pyriformis suspensions of defined cell density were exposed for 1h to different concentrations of the test item. Fluorescein diacetate (FDA) was added and after 30 min fluorescein was measured fluometrically to assess the in vivo metabolic activity of cells as indication of viability.

Flask technique:
T. pyriformis suspensions of defined cell density were cultured in 500 ml Fernbach flasks and exposed to various concentrations of the test item for 9h. Every hour (beginning immediately after exposure start) an aliqout was taken from the suspension and cell density counted. Toxic effects were characterized by determination of generation time, i.e. doubling time.

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): deionized water

Test organisms (species):

Tetrahymena pyriformis

Details on inoculum:

TEST ORGANISM
- Common name: Tetrahymena pyriformis
- Strain: GL

Cell culture:
The ciliated protozoan T. pyriformis, strain GL, was cultured axenically at 28°C in the proteose-peptone yeast salts (PPYS)-defined medium containing 2% proteose-peptone (Difco,Detroit, MI, USA), 0.1% yeast extracts (Difco), and inorganic salts (according to Plessner P, Rasmussen L, Zeuthen E. 1964. Techniques used in the study of synchronous Tetrahymena. In Zeuthen E, ed, Synchrony in Cell Division and Growth. Intersciences, New York, NY, USA, pp 534–565). The cells were maintained in the exponential growth phase.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

9 h

Remarks on exposure duration:

Referring to flask method; exposure duration for FDA method: 1h

Test temperature:

25°C (FDA method)/ 28°C (Flask technique)

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 ml Fernbach flask (Flask technique)
- No. of organisms per vessel: initially 1000 cells (FDA method)/ 100000 (Flask technique)
- No. of vessels per concentration (replicates): 6-8 (FDA method)/ no data (Flask technique)
- No. of vessels per control (replicates): 6-8 (FDA method)/ no data (Flask technique)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Volvic water

OTHER TEST CONDITIONS
Test was performed in darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
fluorescence of fluorescein as surrogate of cell viability (FDA method)
genereation time (Flask technique)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Reference substance (positive control):

not specified

Duration:

1 h

Dose descriptor:

EC50

Effect conc.:

26.95 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: non-specific esterase activity

Remarks on result:

other: SD = 2.35 mg/L

Duration:

9 h

Dose descriptor:

IC50

Effect conc.:

210 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth inhibition

Reported statistics and error estimates:

In the FDA technique, the median effective concentration (EC50) is the concentration of xenobiotics required to induce a 50% decrease in the fluorescence in comparison with the untreated cells. The EC50 values were quantified by regression analysis computed with StatView SE 1.03. The concentrations were transformed into logarithms, and the data were fitted to the following regression model:

Decrease in fluorescence (%) = a + b(log concentration)

In the flask technique, the toxicity of the tested substances was determined by the calculation of the median inhibitory concentration (IC50), which is the concentration of toxicants required to induce a 50% increase in RGT. For each assay, the IC50 was calculated by regression analysis computed with StatView SE 1.03. The data were fitted to the following regression model:

RGT (%) = a + b(concentration)

log mean 9-h IC50 or log mean 30-min EC50 = a + b(log mean 1-h EC50)

Statistical comparison of cellular densities of populations exposed and not exposed to chemical compounds was performed using Student’s t test (p , 0.05).

Validity criteria fulfilled:

not applicable

Conclusions:

The reported EC50(1h) are 26.95 +- 2.35 mg/L mg/L and the IC50(9h) is 210 mg/L.

Executive summary:

In a 1 h / 9 h toxicity study, cultures of Tetrahymena pyriformis were exposed to various concentrations of Mn2+ under static conditions. The EC50 (1h) and the IC50 (9h) values based on the metabolic activity resp. growth inhibition were 26.95 mg/L and 210 mg/L, respectively. This toxicity study was classified as acceptable and reliable with restrictions and satisfies general scientific requirements.

Reference 2

Endpoint:

toxicity to microorganisms, other

Remarks:

DIN 38412, part 8 (Pseudomonas Zellvermehrungshemm-Test)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: basic scientific principles are met; read-across

Qualifier:

according to guideline

Guideline:

DIN 38412-8 (Pseudomonas Zellvermehrungshemmtest)

Deviations:

not specified

GLP compliance:

not specified

Specific details on test material used for the study:

No details provided.

Analytical monitoring:

not specified

Details on sampling:

No details provided.

Vehicle:

not specified

Details on test solutions:

No details provided.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

No details provided.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

16 h

Post exposure observation period:

No details reported.

Hardness:

No details provided.

Test temperature:

21°C

pH:

No details provided.

Dissolved oxygen:

No details provided.

Salinity:

Not applicable.

Nominal and measured concentrations:

No details provided.

Details on test conditions:

TEST CONCENTRATIONS
- Test concentrations: 3 range of concentration tested: 0-40 mg/L; 80-5000 mg/L; > 5000 mg/L

Reference substance (positive control):

not specified

Duration:

16 h

Dose descriptor:

EC0

Effect conc.:

> 5 000 mg/L

Nominal / measured:

not specified

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

0-40 mg/L: no effect
80-5000 mg/L: stimulation of growth
> 5000 mg/L: no stimulation of growth but not toxic

Results with reference substance (positive control):

No details given.

Reported statistics and error estimates:

No details given.

Validity criteria fulfilled:

not specified

Remarks:

Only limited information available.

Conclusions:

EC0 (16 h) > 5000 mg/L

Executive summary:

The toxicity of Sodium gluconate (CAS 527-07-1) was tested according to the German standard procedure DIN 38412, part 8 (Pseudomonas Zellvermehrungshemm-Test). As test organism, Pseudomonas putida has been used. Three different concentration ranges were tested: 0 -40 mg/L, 80 -5000 mg/L and > 5000 mg/L. It was reported that in the 0 -40 mg/L concentration range, no effect were observed. In the 80 -5000 mg/L concentration range, growth was stimulated. For the range of > 5000 mg/L, neither stimulation of growth nor toxic effects were observed. As final result, the EC0 (16 h) amounts to > 5000 mg/L.

Reference 3

Endpoint:

toxicity to microorganisms, other

Remarks:

mortality and malformations in Spirostomum ambiguum

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test was not performed according standard guideline, no data on corresponding cation or raw data was given. However, the provided data indicate that the test was well-performed and satisfies general scientific requirements.

Qualifier:

no guideline available

Principles of method if other than guideline:

Spirostomum ambiguum were placed in a 24 well multiwell plate, 10 protozoae / well. They were exposed in darkness over a period of 24 h and 48 h to various concentrations of the test item in triplicates. Two kinds of test responses were observed: (1) different deformations (morphological changes) and (2) lethal response. On this basis two values were calculated for each row of the microplate:
EC50: the concentration producing different deformations of 50% of the test organisms
LC50: the concentration producing lethal response of 50% of the test organisms

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Each test was one control and five toxicant’s concentrations with three duplicates per concentration.
Dilution of the sample (logarithmic progression) was made directly in the plate. For this purpose, the following amount of diluent and sample was added to all four cells in each column: 0, 0.44, 0.68, 0.82, 0.90, 1.00 ml of diluent and 1.00, 0.56, 0.32, 0.18, 0.10, 0 ml of sample.

Test organisms (species):

other: Spirostomum ambiguum

Details on inoculum:

TEST ORGANISM
- Common name: Spirostomum ambiguum
- Source: The strain was originally collected in Kampinos National Park near Warsaw and has been cultured in laboratory for more than 20 years
- Culturing the Spirostomum ambiguum: S. ambiguum was routinely cultured in 5-1 aquariums containing 4 L of natural, unpolluted water originating from a very deep source (pH = 7.5; total hardness 150 mg CaCO3/L). Cultures were maintained at 20–25°C in darkness. Every 4 weeks, two-thirds of the water in the aquarium was replaced with fresh water.

ACCLIMATION
- Type and amount of food: flaked oats and dried alder leaves (50:1)
- Feeding frequency: once / week

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Protozoan were observed after 24h and 48h

Hardness:

150 mg CaCO3/L

Test temperature:

25°C

pH:

7.5

Details on test conditions:

TEST SYSTEM
- Test vessel: 24 well (6 x 4) polystyrene multiwell plate
- No. of organisms per vessel: 10 cells per well
- No. of vessels per concentration (replicates): 3 wells
- No. of vessels per control (replicates): 3 wells

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tyrod solution

OTHER TEST CONDITIONS
- Adjustment of pH: water had a pH = 7.5
- Photoperiod: 24h darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Two kinds of test responses were observed after 24h and 48h: (1) different deformations, which means morphological changes such as shortening, bending of the cell, and so forth; and (2) lethal response (L), spherical deformation and autolysis. On this basis two values were calculated for each row of the microplate:
EC50: the concentration producing different deformations of 50% of the test organisms, and LC50: the concentration producing lethal response of 50% of the test organisms.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: test solutions were prepared with 0, 0.44, 0.68, 0.82, 0.90, 1.00 ml of diluent and 1.00, 0.56, 0.32, 0.18, 0.10, 0 ml of sample
- Range finding study: yes
- Test concentrations: serial dilution (2X)
- Results used to determine the conditions for the definitive study: Concentrations to the definitive test were then chosen: between 0 and 100% lethality of S. ambiguum.

Reference substance (positive control):

not specified

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

92.8 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: morphology

Remarks on result:

other: SD = 44.7 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

148 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: mortality

Remarks on result:

other: SD = 15.5 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

109 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: morphology

Remarks on result:

other: SD = 66.3 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

146 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

other: mortality

Remarks on result:

other: SD = 19.5 mg/L

Reported statistics and error estimates:

Mean values (EC50 and LC50) ± SD were calculated for the microplate.

Validity criteria fulfilled:

not applicable

Conclusions:

The test was considered to be reliable with restrictions, and therefore the results can considered to be trustworthy. Since Spirostomum ambiguum serves as an additional species to assess the possible hazard of the test item to the environment, the same criteria for classification or non-classification should apply as for algae and other aquatic plants.
EC50 values of 92.8 +/- 44.7 mg/L and 109 +/- 66.3 mg/L are obtained after 24h and 48h, respectively. The reported LC50(24h) is 148 +/- 15.5 mg/L, the LC50(48h) is 146 +/- 19.5 mg/L.

Executive summary:

In a 24 h / 48 h toxicity study, ciliated protozoa (Spirostomum ambiguum) were exposed to various concentrations of Mn2+ under static conditions. EC50 values of 92.8 +/- 44.7 mg/L and 109 +/- 66.3 mg/L are obtained after 24h and 48h, respectively. The reported LC50(24h) is 148 +/- 15.5 mg/L, the LC50(48h) is 146 +/- 19.5 mg/L.

This toxicity study was classified as acceptable and reliable with restrictions and satisfies general scientific requirements.

Description of key information

The toxicity to microorganisms is attributed to the metal ion and not the (readily biodegradable) glucoheptonate ion. Therefore, effect concentrations for the manganese salts are way lower than the ones determined for sodium gluconate and should be considered for the safety assessment.

According to ECHA REACH Guidance Chapter R.7b (v4.0; July 2017), data from ciliate growth inhibition tests, preferably with the speciesTetrahymena are considered relevant for the risk assessment for STPs According to an international pilot ring test, a growth test with the ciliate Tetrahymena pyriformis was recommended for ecotoxicological risk assessment by the German Federal Environmental Agency. Furthermore, short-term measurements in the order of hours are preferred, in accordance with the hydraulic retention time in a STP (e.g. 10 h).

Therefore, the study performed by Bogaerts et al. (1998) is considered as most relevant for the PNECstp derivation and the IC50 (9h) value of 1633.5 mg/L serves as key value for the chemical safety assessment.

Key value for chemical safety assessment

EC50 for microorganisms:

1 633.5 mg/L

Additional information

There is no data available for the target substance manganese glucoheptonate on toxicity towards microorganisms. However, there is data available for the source substances Sodium gluconate, manganese sulphate and manganese dichloride which is evaluated in a weight of evidence approach.

Sodium gluconate (SIDS, 2004)

The toxicity of sodium gluconate (CAS 527-07-1) was tested according to the German standard procedure DIN 38412, part 8 (Pseudomonas Zellvermehrungshemm-Test; OECD SIDS, 2004). As test organism, Pseudomonas putida has been used. Three different concentration ranges were tested: 0 -40 mg/L, 80 -5000 mg/L and > 5000 mg/L. It was reported that in the 0 -40 mg/L concentration range, no effect were observed. In the 80 -5000 mg/L concentration range, growth was stimulated. For the range of > 5000 mg/L, neither stimulation of growth nor toxic effects were observed. As final result, the EC0 (16 h) amounts to > 5000 mg/L.

Manganese salts

Data on the toxicity of manganese to microorganisms was reviewed. Toxicity data of manganese on three different species of protozoae is available. The data on manganese compounds allows estimating a corresponding EC for manganese glucoheptonate providing that no toxicity is attributed to the glucoheptonate ion, that the absorption of manganese from this manganese compounds is 100 % and all manganese became systemically available. The reported effect concentrations for the test substances were converted to the registered substance under consideration of the molecular weight and the purity (table 1).

The aim of the study by Klimek et al. (2013) was to assess the toxicity of manganese to the rotifer Lecane inermis, a species tested as a potential biological tool to control activated sludge bulking caused by overgrowth of filamentous bacteria in wastewater treatment plants. LC50 (24 h) value for elemental Mn (2 +) was determined to be 1.9194 mg/L. This corresponds to a LC50 (24 h) value of 15.85 mg MnGHA/L.

In a 1 h / 9 h toxicity study by Bogaerts et al. (1998), cultures of Tetrahymena pyriformis were exposed to various concentrations of Mn2+ under static conditions. The EC50 (1h) and the IC50 (9h) values based on the metabolic activity resp. growth inhibition were 26.95 mg/L and 210 mg/L, respectively. The reported EC50(1h) corresponds to a value of 209.6± 18.3 mg/L and the IC50(9h) is 1633.5 mg/L for Mn glucoheptonate.

In a 24 h / 48 h toxicity study, ciliated protozoa (Spirostomum ambiguum) were exposed to various concentrations of Mn2+ under static conditions (Nalecz-Jawecki et al., 1998). EC50 values based on morphology of 92.8 +/- 44.7 mg/L and 109 +/- 66.3 mg/L are obtained after 24h and 48h, respectively. The reported LC50(24h) is 148 +/- 15.5 mg/L, the LC50(48h) is 146 +/- 19.5 mg/L. Converted to the target substance manganese glucoheptonate the 24h and the 48h EC50s are 721.8±347.7 mg/L and 847.8±515.7 mg/L, respectively. The reported LC50 correspond to a LC50(24h) of 1151.2±120.6 mg/L and a LC50(48h) of 1135.6±151.7 mg/L.

Table 1: Effect concentrations (EC), Lethal concentrations (LC) and inhibitory concentration (IC) derived from studies performed with various manganese compounds and converted to manganese glucoheptonate (MnGHA).

Species	Duration of exposure	Dose descriptor	Mn GHA (70%) mg/L	water media type	Reference
Spirostonum ambiguum	24h	EC50	721.8 ± 347.7	freshwater	Nalecz-Jawecki & Sawicki (1998)
Spirostonum ambiguum	48h	EC50	847.8 ± 515.7	freshwater	Nalecz-Jawecki & Sawicki (1998)
Spirostonum ambiguum	24h	LC50	1151.2 ± 120.6	freshwater	Nalecz-Jawecki & Sawicki (1998)
Spirostonum ambiguum	48h	LC50	1135.6 ± 151.7	freshwater	Nalecz-Jawecki & Sawicki (1998)
Tetrahymena pyriformis	1h	EC50	209.6 ± 18.3	freshwater	Bogaerts et al (1998)
Tetrahymena pyriformis	9h	IC50	1633.5	freshwater	Bogaerts et al (1998)
Lecane inermis	24 h	LC50	15.85	freshwater	Klimek et al. (2013)