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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984a
Deviations:
yes
Remarks:
Test concentrations were not provided.
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
All tests were performed with pH adjusted ISO-medium containing 2 mM CaCl2 , 0.5 mM MgSO4 and 0.078 mM KCI.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP 278/4, Ambleside, UK)
- Method of cultivation: P. subcapitata starter cultures were grown in standard OECD-medium (OECD, 1984a, which has the same composition as described by ISO, 1989) at 20 ± 1 °C with continuous light (5000 lux) and continuous aeration (filtered air).
- Any deformed or abnormal cells observed: none reported. Each week, cultures were visually inspected for contamination using a light microscope.
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Remarks on exposure duration:
After 24, 48 and 72 h of exposure the number of algal cells was counted using a coulter counter.
Test temperature:
20 ± 1 °C
pH:
7.4
Nominal and measured concentrations:
Logarithmic concentration series (1 control + 5 concentrations); no exact values given.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: capped with an air-permeable stopper
- Size, fill volume: 100 mL, 50 mL
- Initial cells density: 1.00E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Adjustment of pH: daily
- Photoperiod: continuous illumination
- Light intensity and quality: 4000 lux
EFFECT PARAMETERS MEASURED (with observation intervals): EC50. 24, 48 and 72 h.
- Determination of cell concentrations: coulter counter.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: logarithmic


Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.191 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Zn2+
Basis for effect:
biomass
Remarks on result:
other: at pH 6
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.137 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Zn2+
Basis for effect:
biomass
Remarks on result:
other: at pH 7
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.071 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Zn2+
Basis for effect:
biomass
Remarks on result:
other: at pH 8
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.133 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Zn2+
Basis for effect:
biomass
Remarks on result:
other: average of three pH
Details on results:
- Exponential growth in the control (for algal test): no information
Reported statistics and error estimates:
EC50s based on biomass were calculated with a logistic model (Haanstra et al., 1985) fitted to the observed area under the growth curve (A) versus measured dissolved metal concentrations (fitting performed with Sigmaplot software, SPSS Inc., Chicago, IL, USA).
Validity criteria fulfilled:
yes
Remarks:
According to the authors, all conditions for the validity of the toxicity tests were fulfilled as prescribed in the standard test procedures (OECD, 1984a; OECD, 1998).
Conclusions:
The 72 h EC50 of zinc added as zinc chloride was 0.133 mg Zn2+/L in average in Pseudokirchneriella subcapitata.
Executive summary:

In the study by De Schamphelaere et al. (2004), the toxicity of Zn2+ towards Pseudokirchneriella subcapitata was examined.The tests were conducted in accordance with OECD Guideline No. 201 (OECD, 1984a). Zinc was added as zinc chloride.

72-h toxicity assays were conducted in OECD-medium. Tests were conducted at pH 6, 7 and 8 with daily manual pH adjustment with HCl. Test media were used as the dilution water to make a logarithmic concentration series (1 control + 5 metal concentrations) of zinc, but precise values were not provided. The assays were conducted in triplicates. The number of cells after 24, 48 and 72 h of exposure was determined and EC50s based on biomass were calculated. No abnormalities were reported.

According to the authors, all conditions for the validity of the toxicity tests were fulfilled as prescribed in the standard test procedures (OECD, 1984a; OECD, 1998).

For zinc, the 72-h EC50s were, in order of increasing pH (pH 6, 7 and 8), 0.191, 0.137 and 0.071.2 mg/L which corresponds to an average of 0.133 mg Zn2+/L.

The average 72 h EC50 of zinc added as zinc chloride was 0.133 mg Zn2+/L in Pseudokirchneriella subcapitata.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
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: toxicity tests were performed in polystyrene microplates for 4 days.
- Short description of test conditions: please refer to section “any other details on materials and methods incl. tables”.
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata NIVA-CHL1
- Age of inoculum (at test initiation): 5 days
Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h
pH:
7.2
Nominal and measured concentrations:
320, 100, 32, 10, 3.2, 1.0, 0.32, 0.1, 0.032, 0.01, 0.0032 and 0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: microplates containing 6 × 4 flat bottom wells
- Material, size, fill volume: polystyrene, 3 mL, 1.8 mL
- Initial cells density: 1.00E+04
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
GROWTH MEDIUM
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): EC50, 96 h
- Determination of cell concentrations: Coulter Multisizer (Software Level 1.8).
TEST CONCENTRATIONS
- Test concentrations: 320, 100, 32, 10, 3.2, 1.0, 0.32, 0.1, 0.032, 0.01, 0.0032 and 0 mg/L
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.08 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Zn2+
Basis for effect:
biomass
Details on results:
- Any stimulation of growth found in any treatment: Zn2+ at concentrations up to 0.01 mg/L slightly enhanced the growth of algal cells in stock alginate beads.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The percent of variance guideline, proposed by Miller et al. and modified by Abdel-Hamid et al. was used to ascertain whether the differences obtained in growth (algal dry weight) of replicate microplate wells differed significantly. Outliers were rejected according to this guideline. All statistical analyses were performed using proper statistical software STATISTICA Ver. 8 (StatSoft, Inc., 2007, Tulsa, OK, USA).

Zn2+ dose-dependently reduced the growth of algae. No cells were able to grow at Zn concentrations above 0.32 mg/L.

Validity criteria fulfilled:
not applicable
Conclusions:
The 96-h EC50 of Zn2+ towards Pseudokirchneriella subcapitata was 0.08 mg/L in a microplate assay.
Executive summary:

In the publication by Al-Hasawi et al. (2020), the toxicity of zinc to Pseudokirchneriella subcapitata was examined. No guideline was followed. Growth inhibition bioassays were conducted in a static assay using polystyrene microplates containing 6 × 4 flat bottom wells.

Zinc was added as zinc chloride. Test concentrations were 320, 100, 32, 10, 3.2, 1.0, 0.32, 0.1, 0.032, 0.01, 0.0032 and 0 mg/L. Each concentration or control had four replicates. The biomass growth was measured in a Coulter Multisizer. The test duration was 96 hours.

Under the conditions of this study, the 96 h EC50 of zinc in Pseudokirchneriella subcapitata was 0.08 mg Zn2+/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
not all information is provided
GLP compliance:
no
Remarks:
GLP compliance was not reported in this publication.
Analytical monitoring:
yes
Details on sampling:
not specified
Vehicle:
no
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: Desmodesmus subspicatus 86.81 SAG
- Source: Sammlung von Algenkulturen der Universitat Göttingen (SAG, Göttingen, Germany)
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: algae were grown in a medium recommended by OECD (1984). The pH of culture medium was adjusted to 8 using NaHCO3 - HCl (0.1 N). Stock cultures were incubated in 250 ml erlenmeyer flasks containing 100 ml of sterilised OECD medium under a photon irradiance of approximately 120 µE m -2 s-1 (cool white fluorescent tubes) in constant illumination by constant shaking at 100 rpm and temperature 25 ± 2°C.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
25 ± 2 °C
Nominal and measured concentrations:
Nominal: 0, 250, 500, 1000, 2000, 4000 µg/L

.
Details on test conditions:
TEST SYSTEM
- Test vessel: erlenmeyer flask
- Size, fill volume: 250 mL, 100 mL
- Renewal rate of test solution: none
- Initial cells density: 8.00E+04 / mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Photoperiod: permanent illumination
- Light intensity and quality: Cool white fluorescent lighting, 120 µE m-2 s-1
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): EC50, 96 h
- Determination of cell concentrations: neubauer improved counter (Marienfeld, Germany)
TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 0, 250, 500, 1000, 2000, 4000 µg/L
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.258 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Zn2+
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): specific growth rate was 0.644.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Experiments were conducted three times in one plate, and the average and standard deviation were calculated. The toxicity of Zn was expressed as the effective concentration giving 50% reduction (EC50) in the number of cells over 96 h compared to the controls. This was calculated using the U.S. Environmental Protection Agency (U.S. EPA) Probit Analysis Program Version 1.5. The dose response equation was X2 tested with 95% confidence. The data relative to growth and growth rate were analysed by a one-way analysis of variance (ANOVA). All significance levels mentioned in the text are at the P<0.05 level.

The growth of the alga decreased with increasing zinc concentrations. A 96-h exposure of D. subspicatus to 250 and 4000 μg/L caused approximately 11.275-70.588 % reduction in cell numbers.

The 96-h EC 50 value obtained for D. subspicatus was 2257.824 (95% confidence intervals of 1856.212 - 2881.057) μg/L.

No effects on phenotypic plasticity were reported in D. subspicatus.

Zn2+ (µg/L)

Specific growth rate D. subspicatus

0

0.644±0.006

250

0.631±0.007

500

0.593±0.004

1000

0.547±0.008

2000

0.512±0.004

4000

0.374±0.004

 

Validity criteria fulfilled:
no
Conclusions:
The 96-h EC50 of zinc (Zn2+) supplied as zinc chloride was 2.258 mg/L in Desmodesmus subspicatus.
Executive summary:

In the publication by Güçlü and Ertan, 2012, the toxicity of zinc towards Desmodesmus subspicatus was examined. 96 h growth inhibition bioassays were conducted in a static test according to OECD-guideline 201 (OECD, 1984) with acceptable restrictions.

Zinc was added as zinc chloride. The effective concentration range was determined in a range-finding test. Culture media of zinc concentrations 0.25, 0.50, 1.00, 2.00 and 4.00 mg/L were used in the experiment. Control cultures were incubated in the same medium without toxicant. There were three replicates for the control and each of the treatment concentrations. The EC50 was determined after 96 h of exposure.

Under the given conditions, the 96 h EC50 of zinc (Zn2+) was 2.258 mg/L in Desmodesmus subspicatus.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
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: toxicity tests were performed in plastic flasks for 96 h.
- Short description of test conditions: please refer to section “any other details on materials and methods incl. tables”.
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Chlorella vulgaris
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- strain: Chlorella vulgaris
- Source: Carolina Biological Supply Co., Burlington, NC
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
15.5 ± 0.5 °C
pH:
~ 6
Nominal and measured concentrations:
0.0, 1.0, 1.8, 2.4, 3.2 and 10 ppm
Details on test conditions:
TEST SYSTEM
- Test vessel: Falcon tissue culture flasks
- Material, size, fill volume: plastic, 30 mL, 5 mL
- Initial cells density: 1.00E+05 cells per mL
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
GROWTH MEDIUM
- Standard medium used: yes
OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity and quality: 5000 Lx
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): EC50, 96 h
- Determination of cell concentrations: counting chamber
TEST CONCENTRATIONS
- Spacing factor for test concentrations: logarithmic
- Range finding study: yes
- Test concentrations: 0.0, 1.0, 1.8, 2.4, 3.2 and 10 ppm zinc.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Zn2+
Basis for effect:
growth rate
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The resulting data was treated according to the statistical method of probit analysis (Bliss,1952; Finney, 1964a,1964b; and Mather, 1965).
Validity criteria fulfilled:
not applicable
Conclusions:
The 96-h EC50 of zinc (Zn2+) towards Chlorella vulgaris was 2.4 mg/L.
Executive summary:

In the publication by Rachlin and Farran (1974), the toxicity of zinc towards Chlorella vulgaris was examined. No guideline was followed.

96-h toxicity assays were conducted in Bristol’s medium. Tests were conducted approximately at pH 6. Test media were used as the dilution water to make a logarithmic concentration series: 0, 1.0, 1.8, 2.4, 3.2 and 10 mg Zn2+/L. Zinc was added as zinc sulfate.

The number of cells after 96 h of exposure was determined and EC50s based on growth rate were calculated. No information on abnormalities was provided.

Under the conditions of this study, the 96 h EC50 of Zn2+ towards Chlorella vulgaris was 2.4 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
other: EPA/600/4-85/014, P. 76–103. Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms.
Version / remarks:
1985
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Ankistrodesmus falcatus
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- strain: Ankistrodesmus falcatus
- Source (laboratory, culture collection): Starr Collection, University of Texas, USA (UTEX No 749)
- Age of inoculum (at test initiation): 7-10 days
- Method of cultivation: according to American Society for Testing and Materials (ASTM), 1988
- Any deformed or abnormal cells observed: none reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Nominal and measured concentrations:
0.31 – 5.0 mg/L, 5 concentrations plus control
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, fill volume: glass, 125 mL, 25 mL
- Initial cells density: 5.00E+04 cells/mL
- No. of vessels per concentration (replicates): 3
GROWTH MEDIUM
- Standard medium used: yes
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): EC50, 96 h
- Determination of cell concentrations: spectrophotometric at 750 nm
TEST CONCENTRATIONS
- Test concentrations: 0.31 – 5.0 mg/L, 5 concentrations plus control
Reference substance (positive control):
yes
Remarks:
potassium dichromate from 0.4 to 7 mg/L
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.432 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Zn2+
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Zn2+
Basis for effect:
growth rate
Details on results:
No marked toxic effects on A. falcatus growth were observed.
Results with reference substance (positive control):
- EC50 (96 h): 1.05 mg/L
Reported statistics and error estimates:
One-way statistical analysis of variance (p<0.05) was applied to the results obtained. The U.S. Environmental Protection Agency (U.S. The U.S. Environmental Protection Agency (U.S. EPA) Probit analysis (1985) method (PC version 1.4) was employed to calculate the EC50 and the algal growth inhibition in respect to the control value at 96 h with 95% confidence intervals. Dunnett’s procedure (Dunnett, 1955) was used to calculate the no observable effect concentration (NOEC) value (U.S. EPA, 1985).

The most commonly used alga S. capricornutum appears very sensitive to heavy metals when compared with A. falcatus.

Validity criteria fulfilled:
no
Conclusions:
The 96-h EC50 of Zn2+ in Ankistrodesmus falcatus was 2.432 mg/L.
Executive summary:

In the publication by Magdaleno et al. (1997), the toxicity of Zn2+ towards Ankistrodesmus falcatus was examined according to a U.S. EPA guideline (1985). Not all details on the experimental procedure are reported.

96-h toxicity assays under static conditions were conducted in ASM-1 nutrient medium without EDTA. Zinc was added as ZnSO4 x 7H2O in 5 concentrations (0.31 – 5.0 mg/L) plus medium control.

The number of cells after 96 h of exposure was determined and EC50s based on growth rate were calculated. No abnormalities were reported. The authors state that the most commonly used alga Pseudokirchneriella subcapitata appears very sensitive to heavy metals when compared with Ankistrodesmus falcatus.

Under the conditions of this study, the 96-h EC50 of Zn2+ in Ankistrodesmus falcatus was 2.432 mg/L. The corresponding 96 -h NOEC was 0.31 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
72 h growth inhibition bioassays were conducted in a static test using polystyrene FB microplates with 96 wells. For further information, please see section "any other details on materials and methods incl. tables".
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Strain: Pseudokirchneriella subcapitata (Raphidocelis subcapitata) SKULBERG 1959/1
- Source (laboratory, culture collection): Culture Collection of Autotrophic Organisms, Institute of Botany, Academy of Sciences of the Czech Republic (CCALA)
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The green algae were pre-cultivated in ISO medium under continuous cool white light luminescent tubes of an intensity of 6000 lux. Cultures were continuously shaken at 100 rpm at 27 ± 1 °C.
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Details on test conditions:
TEST SYSTEM
- Test vessel: FB microplates with 96 wells
- Material, fill volume: polystyrene, 0.25 mL
- Initial cells density: 8.00E+04 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
GROWTH MEDIUM
- Standard medium used: yes
EFFECT PARAMETERS MEASURED: EC50, 72 h
- Determination of cell concentrations: biomass growth was measured as optical density at 680 nm by a spectrophotometer for micropIates (Sumal PE-2, Carl Zeiss, Jena).
TEST CONCENTRATIONS
- Range finding study: yes

Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.799 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
ZnSO4
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.324 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Zn2+
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): no information given
- Observation of abnormalities (for algal test): none reported
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The EC50 values were determined from the polynomial curve fitted in the data by the least squares method.

In total, 7 algal species were tested. The tested species (apart from Pseudokirchneriella subcapitata) and their respective EC50-values for ZnSO4 are summarized in the table below:

Species

Duration [h]

EC50 ZnSO4 [mg/L]

Scenedesmus quadricauda

72

0.813

Desmodesmus subspicatus

72

0.767

Chlamydomonas reinhardtii

72

2.171

Synechococcus leopoliensis

96

10.1

Stichococcus bacillaris

72

0.724

Chlorella kessleri

72

2.305

 

Validity criteria fulfilled:
not applicable
Conclusions:
The 72 h EC50 of zinc sulfate in Pseudokirchneriella subcapitata was 0.799 mg/L or 0.324 mg Zn2+/L, respectively.
Executive summary:

In the publication by Rojíčková-Padrtová and Maršálek, 1999, the toxicity of zinc sulfate towards Pseudokirchneriella subcapitata was examined. 72 h growth inhibition bioassays were conducted in a static test using polystyrene FB microplates with 96 wells. No guideline was followed, but the basic requirements are similar to OECD guideline 201.

Test concentrations were chosen according to a range-finding study, but precise values are not reported. Each concentration or control had six replicates. The biomass growth was measured by a spectrophotometer for microplates. The test duration was 72 hours.

Under the conditions of this study, the 72 h EC50 of zinc sulfate in Pseudokirchneriella subcapitata was determined to be 0.799 mg/L or 0.324 mg Zn2+/L, respectively.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
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.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across hypothesis is based on transformation of the target and source substances to common compounds (scenario 1 of the RAAF). The target substance zinc monoglycinate sulfate and the source substances zinc sulfate and zinc bisglycinate consist of the Zn2+ cation and the respective anion. The amino acid glycine is constituent of both the target substance zinc monoglycinate sulfate and the source substance zinc bisglycinate.
It is generally accepted that the Zn2+ cation (as measure for dissolved zinc species) is the determining factor for toxicity and ecotoxicity, but not sulfate or glycine.
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance zinc monoglycinate sulfate is a chelate-complex which consists of the divalent zinc ion as centre-ion and glycine as ligand. The remaining sulfate group stabilizes the center ion within the complex.
Zinc monoglycinate sulfate and the source substance zinc sulfate are ionic and consist of the Zn2+ cation and the respective anions. It is generally accepted that the zinc cation is the determining factor for toxicity and ecotoxicity. Therefore, this read-across approach is based on the assumption that the metal cation of both the target and the source substance, zinc, is the relevant component for assessment of toxicity and ecotoxicity.
The anion of the target substance is the essential amino acid glycine and the sulfate anion. In the source substance, it is the sulfate anion. These anions are not considered as (eco)toxicologically relevant at the given concentrations.
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
3. ANALOGUE APPROACH JUSTIFICATION
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
4. DATA MATRIX
Please refer to the justification for read-across analogue approach in Chapter 13.2 for more detailed information.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.52 mg/L
Nominal / measured:
nominal
Conc. based on:
other: calculated for zinc monoglycinate sulfate
Basis for effect:
biomass
Remarks on result:
other: Based on the publication by De Schamphelaere et al. (2004). Average of three pH.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.133 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Zn2+
Basis for effect:
biomass
Remarks on result:
other: De Schamphelaere et al. (2004). A verage of three pH.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.08 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Zn2+
Basis for effect:
biomass
Remarks on result:
other: Al-Hasawi et al. (2020).
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.258 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Zn2+
Basis for effect:
growth rate
Remarks on result:
other: Güçlü and Ertan, 2012
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.432 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Zn2+
Basis for effect:
growth rate
Remarks on result:
other: Magdaleno et al., 1997
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Zn2+
Basis for effect:
growth rate
Remarks on result:
other: Rachlin and Farran, 1974
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.324 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Zn2+
Basis for effect:
growth rate
Remarks on result:
other: Rojíčková and Maršálek, 1999
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
93 748.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Glycine
Basis for effect:
growth rate
Remarks on result:
other: results for ECOSAR Class Aliphatic Amines results from QSAR prediction
Validity criteria fulfilled:
not applicable
Conclusions:
There is no information available on aquatic toxicity of zinc monoglycinate sulfate. However, based on the information available for the constituents glycine and Zn2+, the read-across is based on the hypothesis that metal ions in combination with amino acids do not exhibit higher ecotoxicity than the metal ions alone.
The 48-h LC50 for aquatic algae was determined in various studies. The EC50 value chosen in this approach is 0.133 mg Zn2+/L according to De Schamphelaere et al., 2004. This corresponds to a 48-h LC50 of 0.52 mg zinc monoglycinate sulfate monohydrate/L in Pseudokirchneriella subcapitata.
Executive summary:

There is no information available on aquatic toxicity of zinc monoglycinate sulfate. However, based on the information available for the constituents glycine and Zn2+, the read-across is based on the hypothesis that the metal ion in combination with amino acids does not exhibit higher ecotoxicity than the metal ion alone.


The anions of the target substanceare the essential amino acid glycine and sulfate. These anions are not considered as ecotoxicologically relevant at the given concentrations. Instead, Zn2+ is the ecotoxicologically critical component.
 The zinc EC50 for aquatic algae were determined in various studies. The reported 72- or 96-h EC50 values in reliable studies range from 0.08 to 2.432 mg Zn2+/L. The zinc toxicity in green algae was shown to be strongly dependent on the examined species. The most sensitive species was Pseudokirchneriella subcapitata (72-h EC50: 0.133 mg Zn2+/L), while Ankistrodesmus falcatus (96-h EC50: 2.432 mg Zn2+/L), Chlorella vulgaris (96-h EC50: 2.4 mg Zn2+/L) and Desmodesmus subspicatus (96-h EC50: 2.258 mg Zn2+/L) were less sensitive. 
The EC50 value chosen in this approach is 0.133 mg Zn2+/L according to De Schamphelaere et al. (2004) for the following reasons: the study reported by De Schamphelaere et al. (2004) was conducted according to OECD guideline 201 (RL1) using the sensitive species Pseudokirchneriella subcapitata, for regular 72 hours. In the other studies using Pseudokirchneriella subcapitata by Al-Hasawi et al.(2020)  and Rojíčková-Padrtová and Maršálek (1999), no guideline was followed and the study duration was 96 h. 
 The 72-h EC50 value chosen in this approach is 0.133 mg Zn2+/L.
  This corresponds to a 72-h EC50 of 0.52 mg zinc monoglycinate sulfate monohydrate/L in Pseudokirchneriella subcapitata.

Description of key information

- Key study: De Schamphelaere et al., 2004: according to OECD 201, RL1. The average 72 h EC50 of zinc added as zinc chloride was 0.133 mg Zn2+/L in Pseudokirchneriella subcapitata. This corresponds to a 72-h EC50 of 0.52 mg zinc monoglycinate sulfate monohydrate/L in Pseudokirchneriella subcapitata.


- supporting study: Rojíčková-Padrtová and Maršálek, 1999, no guideline followed, RL2: the 72 h EC50 of zinc sulfate in Pseudokirchneriella subcapitata was 0.799 mg/L or 0.324 mg Zn2+/L, respectively.


- supporting study: Al-Hasawi et al., 2020: no guideline followed, RL2: the 96 h EC50 of zinc added as zinc chloride in Pseudokirchneriella subcapitata was 0.08 mg Zn2+/L.


- supporting study: Güçlü and Ertan, 2012: OECD guideline 201, RL2. The 96 h EC50 of zinc (Zn2+) added as zinc chloride was 2.258 mg/L in Desmodesmus subspicatus.


- supporting study: Magdaleno et al., 1997: U.S. EPA guideline (1985), RL2: the 96-h EC50 of Zn2+ added as ZnSO4 x 2H2O in Ankistrodesmus falcatus was 2.432 mg/L. The corresponding 96 -h NOEC was 0.31 mg/L.


- supporting study: Rachlin and Farran, 1974: no guideline followed, RL2: the 96 h EC50 of Zn2+ added as ZnSO4 in Chlorella vulgaris was 2.4 mg/L.


- QSAR prediction, RL2: glycine does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).


The EC50 value chosen in this approach is 0.133 mg Zn2+/L according to De Schamphelaere et al. (2004) for the following reasons: the study reported by De Schamphelaere et al. (2004) was conducted according to OECD guideline 201 (RL1) using the sensitive species Pseudokirchneriella subcapitata, for regular 72 hours. In the other studies using Pseudokirchneriella subcapitata by Al-Hasawi et al.(2020)  and Rojíčková-Padrtová and Maršálek (1999), no guideline was followed and the study duration was 96 h. 

Key value for chemical safety assessment

EC50 for freshwater algae:
0.52 mg/L

Additional information

In the study by De Schamphelaere et al. (2004), the toxicity of Zn2+ towards Pseudokirchneriella subcapitata was examined.The tests were conducted in accordance with OECD Guideline No. 201 (OECD, 1984a). Zinc was added as zinc chloride. 72-h toxicity assays were conducted in OECD-medium. Tests were conducted at pH 6, 7 and 8 with daily manual pH adjustment with HCl. Test media were used as the dilution water to make a logarithmic concentration series (1 control + 5 metal concentrations) of zinc, but precise values were not provided. The assays were conducted in triplicates. The number of cells after 24, 48 and 72 h of exposure was determined and EC50s based on biomass were calculated. No abnormalities were reported. According to the authors, all conditions for the validity of the toxicity tests were fulfilled as prescribed in the standard test procedures (OECD, 1984a; OECD, 1998). For zinc, the 72-h EC50s were, in order of increasing pH (pH 6, 7 and 8), 0.191, 0.137 and 0.071.2 mg/L which corresponds to an average of 0.133 mg Zn2+/L. The average 72-h EC50 of zinc added as zinc chloride was 0.133 mg Zn2+/L in Pseudokirchneriella subcapitata.

In the publication by Rojíčková-Padrtová and Maršálek, 1999, the toxicity of zinc sulfate towards Pseudokirchneriella subcapitata was examined. 72 h growth inhibition bioassays were conducted in a static test using polystyrene FB microplates with 96 wells. No guideline was followed, but the basic requirements are similar to OECD guideline 201. Test concentrations were chosen according to a range-finding study, but precise values are not reported. Each concentration or control had six replicates. The biomass growth was measured by a spectrophotometer for microplates. The test duration was 72 hours. Under the conditions of this study, the 72-h EC50 of zinc sulfate in Pseudokirchneriella subcapitata was determined to be 0.799 mg/L or 0.324 mg Zn2+/L, respectively.

In the publication by Al-Hasawi et al. (2020), the toxicity of zinc to Pseudokirchneriella subcapitata was examined. No guideline was followed. Growth inhibition bioassays were conducted in a static assay using polystyrene microplates containing 6 × 4 flat bottom wells. Zinc was added as zinc chloride. Test concentrations were 320, 100, 32, 10, 3.2, 1.0, 0.32, 0.1, 0.032, 0.01, 0.0032 and 0 mg/L. Each concentration or control had four replicates. The biomass growth was measured in a Coulter Multisizer. The test duration was 96 hours. Under the conditions of this study, the 96 h EC50 of zinc in Pseudokirchneriella subcapitata was 0.08 mg Zn2+/L.

In the publication by Güçlü and Ertan, 2012, the toxicity of zinc towards Desmodesmus subspicatus was examined. 96 h growth inhibition bioassays were conducted in a static test according to OECD-guideline 201 (OECD, 1984) with acceptable restrictions. Zinc was added as zinc chloride. The effective concentration range was determined in a range-finding test. Culture media of zinc concentrations 0.25, 0.50, 1.00, 2.00 and 4.00 mg/L were used in the experiment. Control cultures were incubated in the same medium without toxicant. There were three replicates for the control and each of the treatment concentrations. The EC50 was determined after 96 h of exposure. Under the given conditions, the 96 h EC50 of zinc (Zn2+) was 2.258 mg/L in Desmodesmus subspicatus.

In the publication by Magdaleno et al. (1997), the toxicity of Zn2+towards Ankistrodesmus falcatus was examined according to a U.S. EPA guideline (1985). Not all details on the experimental procedure are reported. 96-h toxicity assays under static conditions were conducted in ASM-1 nutrient medium without EDTA. Zinc was added as ZnSO4x 7H2O in 5 concentrations (0.31 – 5.0 mg/L) plus medium control. The number of cells after 96 h of exposure was determined and EC50s based on growth rate were calculated. No abnormalities were reported. The authors state that the most commonly used alga Pseudokirchneriella subcapitata appears very sensitive to heavy metals when compared with Ankistrodesmus falcatus. Under the conditions of this study, the 96-h EC50 of Zn2+ in Ankistrodesmus falcatus was 2.432 mg/L. The corresponding 96 -h NOEC was 0.31 mg/L.

In the publication by Rachlin and Farran (1974), the toxicity of zinc towards Chlorella vulgaris was examined. No guideline was followed. 96-h toxicity assays were conducted in Bristol’s medium. Tests were conducted approximately at pH 6. Test media were used as the dilution water to make a logarithmic concentration series: 0, 1.0, 1.8, 2.4, 3.2 and 10 mg Zn2+/L. Zinc was added as zinc sulfate. The number of cells after 96 h of exposure was determined and EC50s based on growth rate were calculated. No information on abnormalities was provided. Under the conditions of this study, the 96 h EC50 of Zn2+ towards Chlorella vulgaris was 2.4 mg/L.

Glycine: 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.