Hydrogen (glycinato-N,O)[sulphato(2-)-O,O']ferrate(1-)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: A strain of Chlorella vulgaris was cultivated with FeCl3 in the following concentrations: 0, 0,0001, 0.0002, 0.0003, 0.0005, 0.001, 0.002, 0.003, 0.005, 0.0075, 0.01, 0.02, 0.03, 0.05, 0.075, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 24, and 30% (w/v) in mineral medium. The basal medium consisted of: 0.05 % K2HPO4 ; 0.05 %, KH2PO4 ; 0.05 %, (NH4)2SO4; 0.05% KNO3; copper-free distilled water, 20% of which was replaced by activated-carbon-treated tapwater or well-water. Cultivation took place at room temperature in daylight at a window situated on the North. As under these conditions the growth rate was rather low, the first observations, i.e. comparisons with the blank cultures, were made several weeks after inoculation. The observations were continued until the cultures were 3 to 4 months old.
As all metals that produce weak bases may profoundly affect the pH of the culture solutions, the pH tolerance of Chlorella was examined in a separate series of experiments. Culture solutions with pH values within the range of 2.2 - 8 were prepared. After inoculation with C. vulgaris and incubation for several weeks, growth occurred in all flasks except those with a pH of 3.2 or lower. For further exploration of the problem, pH measurements were made of 0.5% solutions of salts of metals forming weak bases.
- Parameters analysed / observed: For every salt examined, the highest concentration tolerated as well as the lowest concentration preventing growth was registered.

GLP compliance:

no

Remarks:

study was conducted prior to implementation of the GLP Guideline

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Fifty ml of the mineral medium described above was brought into 150 mLerlenmeyer flasks. In general the medium was supplemented with 0 (control) 0.0001 - 0.0002 - 0.0003 - 0.0005 - 0.001 - 0.002 - 0.003 - 0.005 - 0.0075 - 0.01 - 0.02 - 0.03 - 0.05 - 0.075 - 0.1 - 0.25 - 0.5 - 1 - 1.5 - 2 - 2.5 - 3 - 3.5 - 4 - 4.5 - 5 - 6 - 7 - 8 - 9 - 10 - 20 - 24 and 30% respectively of the metal salt to be investigated

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: Chlorella vulgaris
- Source (laboratory, culture collection): from the collection of the Laboratory of Microbiology, Technological University, Delft
- Method of cultivation: For inoculation, cells were taken from Chlorella cultures on agar slants; the amount was kept so small that no green tinge could be observed after shaking the inoculated medium.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

4 mo

Remarks on exposure duration:

The alga were incubated for several months instead of short-time exposure e.g. 96h.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): stoppered with cotton plugs
- Material, size, headspace, fill volume: Erlenmeyer falsks of 150 mL fill volume
- Aeration: No
- Initial cells density: cells were taken from Chlorella cultures on agar slants; the amount was kept so small that no green tinge could be observed after shaking the inoculated medium.

GROWTH MEDIUM
- Detailed composition if non-standard medium was used: The basal medium consisted of: 0.05 % K2HPO4 ; 0.05 %, KH2PO4 ; 0.05 %, (NH4)2SO4; 0.05% KNO3; copper-free distilled water, 20% of which was replaced by activated-carbon-treated tapwater or well-water.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no, pH between 2.2 and 8 are well tolerated according to the author
- Photoperiod:Not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Other: For every salt examined, the highest concentration tolerated as well as the lowest concentration preventing growth was registered.

Reference substance (positive control):

no

Key result

Duration:

16 wk

Dose descriptor:

EC0

Effect conc.:

0.003 g/L

Nominal / measured:

nominal

Conc. based on:

other: Fe3+

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

Results were determined for trivalent Iron

Details on results:

- Other: At concentration 0.5% (w/v) the medium became massively acidic (pH 1.5) thus, the inhibitory effect can be regarded to the pH change. Neutralization of the pH would have lead to precipitation and would thus limit the exposure to Fe3+

Validity criteria fulfilled:

not applicable

Conclusions:

In the present study of Dooren De Jong (1965) which was conducted prior to implementation of GLP and OECD guidelines, Chlorella vulgaris algae were exposed to 0 (control) 0.0001 - 0.0002 - 0.0003 - 0.0005 - 0.001 - 0.002 - 0.003 - 0.005 - 0.0075 - 0.01 - 0.02 - 0.03 - 0.05 - 0.075 - 0.1 - 0.25 - 0.5 - 1 - 1.5 - 2 - 2.5 - 3 - 3.5 - 4 - 4.5 - 5 - 6 - 7 - 8 - 9 - 10 - 20 - 24 and 30% (w/v) FeCl3 in mineral medium for several months in order to determine the lowest inhibitory concentration or the highest tolerable concentration respectively. Trivalent iron caused no adverse effects up to a concentration of 0.003 %(w/v) in Chlorella vulgaris under the conditions of the test. However, at concentration 0.5% (w/v) the medium became massively acidic (pH 1.5) thus, the inhibitory effect can be regarded to the pH change. Neutralization of the pH would have lead to precipitation and would thus limit the exposure to Fe3+. Although Fe3+ is the Iron species that is more likely used by organisms it is known to exhibit a higher toxicity presumably based on its physical and chemical properties, i.e. low water solubility and effects on pH.

Executive summary:

In a 4 month toxicity study, cultures of Chlorella vulgaris  were exposed to FeCl3 at nominal concentrations of 0, 0,0001, 0.0002, 0.0003, 0.0005, 0.001, 0.002, 0.003, 0.005, 0.0075, 0.01, 0.02, 0.03, 0.05, 0.075, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 24, and 30% (w/v) in mineral medium.

Trivalent iron caused no adverse effects up to a concentration of 0.003 %(w/v) in Chlorella vulgaris under the conditions of the test. However, at concentration 0.5% (w/v) the medium became massively acidic (pH 1.5) thus, the inhibitory effect can be regarded to the pH change. Neutralization of the pH would have lead to precipitation and would thus limit the exposure to Fe3+.

Although Fe3+ is the Iron species that is more likely used by organisms it is known to exhibit a higher toxicity presumably based on its physical and chemical properties, i.e. low water solubility.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for the algal growth inhibition test in Chlorella vulgaris.

 

Results Synopsis

Test Organism: Chlorella vulgaris

Test Type: n/a

 

4 mo EC0: 0.003 g Fe3+/L

Endpoint(s) Effected:  growth rate

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Soil microalgae, Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11, were exposed to three differnt iron salts, FeSO4, FeCl3 and Fe (NO3)3 in the following concentrations: 0, 5, 10, 15, 20, 25, and 50 mg/L Fe for 96 h. After 96h the aliquots from different treatments were drawn for measuring the cell growth, and cells were counted using Neubauer haemocytometer under the microscope.
- Short description of test conditions: Cells in the exponential growth phase (5 days old) were harvested by centrifugation at 2000×g for 15 min and washed three times with ultrapure water to remove residues of the growth medium and used as the inoculum. Forty-millilitre clear glass tubes containing 10 mL of modified BBM medium (no EDTA and low phosphate) were inoculated with respective inoculum to an initial cell density of 1E+05 cells/mL. Then, the aliquots from iron stock solutions were added to respective treatments. Experiment was carried out in triplicates and the culture tubes were incubated at 25 °C under light (200 μmol photons m²/s) on orbital shaker (120 rpm) for 96 h.
- Parameters analysed / observed: cell growth, chlorophyll amount

GLP compliance:

no

Remarks:

GLP compliance was not reported in this publication

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

other: Three different microalgae were used: Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11

Details on test organisms:

TEST ORGANISM
- Common name: green soil microalgae
- Strain: Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11
- Source (laboratory, culture collection): maintained axenically in the Phycology Laboratory of Global Centre for Environmental Remediation, The University of Newcastle.
- Age of inoculum (at test initiation): Cells in the exponential growth phase (5 days)
- Method of cultivation: Stock cultures were maintained in flasks containing Bold ’ s Basal medium (BBM) under continuous light (200 μmolphotons m²/s PPFD) at 25 ± 2 °C on an orbital shaker. The cultures were periodically checked for any contamination by microscopic analysis as well as by streaking on BBM medium and nutrient agar plates.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Nominal and measured concentrations:

0, 5, 10, 15, 20, 25, 50 mg/L Fe nominal

Details on test conditions:

TEST SYSTEM
- Test vessel:Forty-millilitre clear glass tubes
- Material, size, headspace, fill volume: glass, 10 mL fill volume
- Aeration: No
- Initial cells density: E+05 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: Bold´s Basal Medium
- Detailed composition if non-standard medium was used: detailed composition was not reported

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Not reported
- Photoperiod: Not reported
- Light intensity and quality: 200 µmol photons m²/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber
- Chlorophyll measurement: The total chlorophyll content in the above treatments was analysed in spectrophotometer at 666, 663 and 470 nm using 10-mL aliquots

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Remarks:

Chlorella sp.

Effect conc.:

9.47 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Fe2+

Basis for effect:

growth rate

Remarks on result:

other: 9.47 mg Fe2+/L corresponds to 38.5 mg/L ferrous glycinate

Key result

Duration:

96 h

Dose descriptor:

EC50

Remarks:

Chlamydomonas sp.

Effect conc.:

9.05 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Fe2+

Basis for effect:

growth rate

Remarks on result:

other: 9.05 mg Fe2+/L corresponds to 36.8 mg/L ferrous glycinate

Key result

Duration:

96 h

Dose descriptor:

EC50

Remarks:

Chlorococcus sp.

Effect conc.:

8.49 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Fe2+

Basis for effect:

growth rate

Remarks on result:

other: 8.49 mg Fe2+/L corresponds to 34.5 mg/L ferrous glycinate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No

Reported statistics and error estimates:

Multifactorial ANOVA was carried out to see whether different sources of iron or different algal species or different iron concentrations independently or jointly influence the total chlorophyll content of algae. Similarly, two-way analysis of variance was done to see whether there is a main and interaction effect in iron bioaccumulation among three algae grown in three different iron sources at different time
intervals. All the experiments were carried out in triplicate and data are expressed as mean values ± standard deviation. Two-way ANOVA was done using general linear model and post hoc analysis with Tukey ’ s multiple comparison tests. All statistical analyses were carried out using Minitab 16.1.1 statistical software.

Validity criteria fulfilled:

not applicable

Conclusions:

In the present study of Subramaniyam et al. (2016) the uptake and toxicity of iron was investigated in Chlorella sp., Chlamydomonas sp. and Chlorococcus sp.. The study was conducted for 96h under static conditions and the algae were exposed to the following concentrations of FeSO4 to obtain the divalent iron ion: 0, 5, 10, 15, 20, 25, and 50 mg/L. The EC50 values determined for the three algae species were: 9.47 mg/L, 9.05 mg/L and 8.49 mg/L Fe2+, respectively. These iron concentrations correspond to 38.5 mg/L, 36.8 mg/L, and 34.5 mg/L ferrous monoglycinate sulfate.

Executive summary:

In a 96 hour toxicity study cultures of Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11 were exposed to FeSO₄at nominal concentrations of in Bold´s Basal Medium, under static conditions.

After 96h the aliquots from different treatments were drawn for measuring the cell growth, and cells were counted using Neubauer haemocytometer under the microscope.

Microscopic evaluation of the cells at start and the end of the incubation period revealed no morphological abnormalities.

 

This toxicity study is classified as acceptable and satisfies the requirements for the algal growth inhibition test in Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11.

 

Results Synopsis

Test Organism: Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11

Test Type: Static

 

96 hr EC50: 9.47 Fe2+./L, Chlorella sp.

96 hr EC50: 9.05 Fe2+./L, Chlamydomonas sp.

96 hr EC50: 8.49 Fe2+./L, Chlorococcus sp.

These iron concentrations correspond to 38.5 mg/L, 36.8 mg/L, and 34.5 mg/L ferrous monoglycinate sulfate.

Endpoint(s) Effected:  growth rate

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
The Estimation Programs Interface (EPI) SuiteTM v4.1 contains the ECOlogical Structure Activity Relationship (ECOSAR) software

2. MODEL (incl. version number)
ECOSAR v1.11, June 19, 2002

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS-No.: 56-40-6
SMILES: O=C(O)CN
Log Kow (measured) = at 25°C
Water solubility (measured) = 480 g/L at 20°C
Melting point (measured) = no melting point, decomposition at 325°C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
For further information, please refer to the attached QSAR reporting format

5. APPLICABILITY DOMAIN
For further information, please refer to the attached QSAR reporting format

6. ADEQUACY OF THE RESULT
For further information, please refer to the attached QSAR reporting format

Qualifier:

no guideline followed

Principles of method if other than guideline:

-Software tool(s) used including version: The Estimation Programs Interface (EPI) SuiteTM v4.1 contains the ECOlogical Structure Activity Relationship (ECOSAR) software
- Model(s) used: ECOSAR v1.11
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

93 748.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results for ECOSAR Class Aliphatic Amines

Remarks:

results from QSAR prediction

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

144 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: results for ECOSAR Class Neutral Organic SAR (Baseline Toxicity)

Remarks:

results from QSAR prediction

Validity criteria fulfilled:

not applicable

Remarks:

QSAR prediction

Conclusions:

In the present QSAR prediction conducted with the EPISuite TM; ECOSAR v1.11 software. The program detected two functional groups present in the molecule which are considered to exhibit a more specific mode of toxicity, namely, aliphatic amines and neutral organics moiety. In consideration of the results of the two chemical classes, two LC50 values are estimated. All LC50 values predicted for glycine are far above the 100 mg/L limit concentration. Since the substance falls within the applicability domain of the used model and the predicted values are more than 10-fold greater than the water solubility, no toxic effects are expected to occur up to saturation limits.

Executive summary:

In the present QSAR prediction conducted with the EPISuite TM; ECOSAR v1.11 software. The program detected two functional groups present in the molecule which are considered to exhibit a more specific mode of toxicity, namely, aliphatic amines and amides. In consideration of the results of all three chemical classes, several LC50 values are estimated. The LC50 value predicted for L-glycine is far above the 100 mg/L limit concentration. Since the substance falls in the applicability domain of the used model and the predicted values are more than 10-fold greater than the water solubility, no toxic effects are expected to occur up to saturation limits.

Description of key information

- study conducted with three strains of green microalgae (Subramaniyam, 2016), namely Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11, the algae were exposed to 0, 5, 10, 15, 20, 25, 50 mg/L Fe (from dissolved FeSO4) nominal for 96h under static conditions, EC50 for the three strains were 9.47, 9.05, and 8.49 mg/L Fe+2, respectively. Read-across

- study conducted with Chlorella vulgaris (DoorenDeJong, 1965), Chlorella vulgaris was cultivated with FeCl3 in the following concentrations: 0, 0,0001, 0.0002, 0.0003, 0.0005, 0.001, 0.002, 0.003, 0.005, 0.0075, 0.01, 0.02, 0.03, 0.05, 0.075, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 24, and 30% (w/v) in mineral medium for 4 month, the 4 week EC0 was determined at 0.003g/L corresponding to 3 mg/L Fe3 +. Read-across

- QSAR estimation performed with ECOSAR for glycine, two EC50 values were reported, one for the class aliphatic amines: 93748.5 mg/L and one for neutral organics: 144000 mg/L based on growth rate. Read-across

Key value for chemical safety assessment

EC50 for freshwater algae:

34.5 mg/L

Additional information

There are no data available for the target substance ferrous monoglycinate sulfate regarding the toxicity to aquatic algae and cyanobacteria. However, based on the read-across hypothesis that the target substance dissociates into its constituents, namely Fe²⁺, SO₄^2- and glycine, when it is dissolved in aqueous solutions or is at least enzymatically hydrolysed by the organism, data for the respective constituent was evaluated to determine the aquatic toxicity of the target substance ferrous monoglycinate sulfate.

In the study of Subramaniyam, 2016, three different algae species were exposed to 0, 5, 10, 15, 20, 25, 50 mg/L Fe (from dissolved FeSO4) nominal for 96h under static conditions, EC50 for the three strains were 9.47, 9.05, and 8.49 mg/L Fe2+, respectively. In another study conducted with Chlorella vulgaris (DoorenDeJong, 1965) the algae were exposed to 0, 0,0001, 0.0002, 0.0003, 0.0005, 0.001, 0.002, 0.003, 0.005, 0.0075, 0.01, 0.02, 0.03, 0.05, 0.075, 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 20, 24, and 30% (w/v)dissolved FeCl3 in mineral medium for 4 month, the EC0 for Fe3+ was 3 mg/L.

In order to predict the effects of glycine towards aquatic algae a QSAR estimation was preformed with ECOSAR and revealed an EC50 value of 93748.5 mg/L for aliphatic amines and 144000 mg/L for neutral organics.

Since glycine is an ubiquitously occurring substance which is known to readily metabolised and serves as energy substrate in the intermediary metabolism its toxicity towards aquatic organisms is also considered to be low. Hence, the toxicity of the target substance ferrous monoglycinate sulfate is expected to be determined by Fe2 +. Fe3+ is known to react with other anions and form salts with a low water solubility, thus, its toxicity towards aquatic organisms as reported in the study of DoorenDeJong (1965) which also was conducted over 4 months, a duration which is far above the timeframe recommended by the respective OECD guideline, the EC0 value obtained from this study is only used as supporting information. The EC50 value obtained from the study of Subramaniyam, 2016, is considered as the most relaible value for the estimation of the toxicity of the target substance because the conduction of the study was appropriate to determine an EC50 value with a suitable duration (96h) and under conditions which are similar to that recommended by the OECD guideline 201. The most sensitive algae species appeared to be Chlorococcum sp. with an EC50 value of 8.49 mg Fe2 +/L which corresponds to 34.5 mg ferrous monoglycinate sulfate/L.