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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:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
No clear derivation of effect concentrations (extrapolation)
Principles of method if other than guideline:
The effect of the test substance on the algae was evaluated by measuring changes in chlorophyll content of the algal supensions every 24 h until 72 h of expsoure.
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
not specified
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A typical test tube contained 15 mL of inorganic culture medium, a predetermined amount of the test substance and 5 mL of the algal culture.
- Controls: yes
Test organisms (species):
Chlorella pyrenoidosa
Details on test organisms:
TEST ORGANISM
- Common name: freshwater green algae
- Strain: Emerson, bacteria-free
- Source: in-house culture
- Method of cultivation: A culture was maintained under steady-state conditions.

ACCLIMATION
- Acclimation period: All tests involved a 2 h adaptation period.
- Culturing media and conditions: same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
25 ± 1 °C
pH:
7.0
Nominal and measured concentrations:
Nominal: control, 100, 250, 500, and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Test tubes
- Type (delete if not applicable): Closed with cotton plugs.
- Fill volume: 20 mL plus a predetermined amount of the test substance.
- Aeration: Yes, a stream of 5% CO2 in air gas mixture was supplied to provide the inorganic carbon source and also to keep the algal cells in suspension.
- Initial cells density: 2 mg (dry wt.) equivalent to a packed cell volume of 7.6 mm3 or a chlorophyll content of 0.076 mg.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: Knop´s solution, including the Hutner-EDTA microelement addition (Myers, 1950)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: Prior to each test the test tubes, aluminum cover caps, caillary glass tubes used to supply the air-CO2 mixture, cotton plugs, rubber tubing, glassware and pipettes were sterilized with the aid of an autoclave. Upon cooling, the culture medium, organic solution, and algae suspension were carefully added to each test tube. While the culture medium was not sterilized, studies indicated that the data were not influenced adversely by the use of this technique.
- Photoperiod: continuous illumination
- Light intensity and quality: four 200 W fluorescent lamps with attached aluminum reflectors

EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: spectrophotometer (Beckman, Model DB), according to MacKinney (1941) and Arnon (1949), using a wavelength of 652 µm.
- Other: Photosynthetic gas exchange (oxygen production) was also measured by a direct manometric technique.
Reference substance (positive control):
not specified
Key result
Duration:
72 h
Dose descriptor:
EC100
Effect conc.:
500 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: chlorophyll [mg/L]
Remarks on result:
other: extrapolated value
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Test duration was only 48 h instead of min. 72 h as recommended in the ECHA Guidance R.7b (ECHA, 2016)
Qualifier:
according to
Guideline:
other: DIN 38 412, Part 9 (draft standard)
GLP compliance:
no
Analytical monitoring:
yes
Vehicle:
not specified
Remarks:
In total, 68 substances were tested. In some cases it was necessary to dissolve the substance in a relatively high concentration in pure ethanol (unmethylated, 40 mg in 10 mL) so that only volumes in the µL range were necessary for the stock solution.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: In total 68 substances were tested. The test solutions were prepared in two different ways, depending on the properties of the substance. Water-soluble substances were quantitatively dissolved to produce an optically clear stock solution in 800 mL double-distilled water using magnetic stirrers (maximum 24 h). For very poorly soluble substances, an attempt was also made to dissolve them quantitatively in 800 mL double-distilled water using magnetic stirrers. Optically unclear solutions were filtered through fibre-glass filters and the filtrate was quantified chemically. In some cases it was necessary to dissolve the substance in a relatively high concentration in pure ethanol (unmethylated) (40 mg in 10 mL) so that only volumes in the µL range had to be transferred to the double-distilled water for the production of the stock solution. Before beginning the test, the stock solutions of the substance to be tested (sample) were adjusted to pH 8.0 ± 0.3.
- Chemical name of vehicle: unmethylated pure ethanol (if applicable)
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: freshwater green algae
- Strain: 8681 SAG
- Source: in-house submerged culture
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: 100 mL Erlenmeyer flasks with metal caps containing 20 mL nutrient solution were sterilized in a steam sterilizer for 30 min on 2 consecutive days. After cooling, the contents of each flask were inoculated with 2 mL cell suspension taken from a 10 d old stock culture. The inoculated flasks were placed on a white surface, protected from daylight and exposed to constant lighting from two parallel fluorescent Osram 40 W/30 tubes (distance from each tube 60 cm: irradiance E0, Sy = 24.9 W/m²) at 24 ± 1 °C and relative humidity of 50%. To maintain the test strain, fresh stock cultures were prepared in 10 d intervals.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
- Preparation of preliminary cultures for test: The cultivation of the preliminary cultures was undertaken 3 d prior to the preparation of the test solution. For this purpose, 50 mL nutrient solution (for test cultures) were filled into 300 mL Erlenmeyer flasks with metal caps and inoculated with algal cells from 7 d-old stock cultures. The cell concentration in the preliminary culture flasks was 1E+04/mL. This was to ensure that the culture was still in a process of logarithmic growth after 72 h. The cell material of the preliminary cultures was used after 72 h to inoculate the dilution preparation after the cell concentration had been fixed at 1E+05/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
24 ± 1 °C
pH:
8.0 ± 0.3 (test substance stock solution before test begin)
8.2 (after 24 h), 9.3 (after 72 h)
Nominal and measured concentrations:
Nominal: 1.6 - 200 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks or 250 mL wide-necked (34/35) bottles (the latter for volatile and/or strongly smelling substances)
- Type: closed with metal caps (300 mL Erlenmeyer flasks); closed with ground-glass stoppers (250 mL wide-necked bottles)
- Material, size, headspace, fill volume: Fill volume: 50 mL
- Initial cells density: 1E+04 cells/mL (300 mL flasks) and 1E+05 cells/mL (250 mL bottles)
- Control end cells density: 3E+05 cells/mL (48 h)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: not specified
- Detailed composition if non-standard medium was used: Nutrient solution (for stock cultures): Dissolve in double-distilled water: 496 mg sodium nitrate, NaNO 3, AR; 39 mg dipotassium hydrogen phosphate, K2HPO4 anhydrous, high purity; 75 mg magnesium sulphate, MgSO4.7 H2O, AR; 36mg calcium chloride, CaCI2.2H2O, AR; 40 mg sodium metasilicate, Na2SiO3;
58 mg sodium carbonate, Na2CO3, anhydrous, AR; 3 mg citric acid, C6H8O7.H20, AR; 3 mg iron (IlI) citrate, C6H5FeO2.5H2O; 10 mg disodium salt of ethylene diamine tetraacetic acid, C100H14N2Na2O8. 2H2O. Add 10 mL of the trace elements operating solution, complete to 1 L with double distilled water.
Stock solution (trace elements): Dissolve in double-distilled water: 2.86g boric acid H3BO3, AR; 1.81 g manganese (II) chloride, MnCl2.4H2O. AR; 220 mg zinc sulphate, ZnSO4.7H2O, AR; 80 mg copper (II) sulphate, CuSO4.5H2O, AR; 24 mg sodium molybdate, Na2MoO4.2H2O, AR; 40 mg cobalt (II) chloride, CoCl2.6H2O, AR. Complete solution with double-distilled water to 1 L in a volumetric flask.
Operating solution of trace elements: Complete 4 mL of stock solution with double-distilled water to 100 mL in a volumetric flask.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: double-distilled water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, the stock solutions of the test substance was adjusted to pH 8 ± 0.3 prior to test begin.
- Photoperiod: constant lighting
- Light intensity and quality:irradiance E = 17.0 W/m², Osram L 25/40 fluorescent tubes
- Intervals of water quality measurement: The pH-value was measured in each test and control vessel after determining the optical density.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: biomass (optical density, i.e. turbidity) measured by spectrophotometer (6115 S, filter 578 nm, 1 or 2 cm cell flask)
- Other: monochromatic radiation (578 nm)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
85 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
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:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1981
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
other: Selenastrum capricornutum and Chlorella vulgaris
Details on test organisms:
TEST ORGANISM
- Common name: freshwater green algae
- Source: National Institute for Environmental Studies, Tsukuba, Japan (S. capricornutum); Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan (C. vulgaris)
- Age of inoculum (at test initiation): 4 - 5 d
- Method of cultivation: Precultures were incubated at 21 ± 1 °C in the culture medium Gorham (pH 7.5) under continuous illumination of 4000 lux.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
21 ± 1 °C
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile Erlenmeyer flasks
- Type: foam-plugged
- Material, size, headspace, fill volume: Size: 500 mL; Fill volume: 100 mL; Headspace: 400 mL
- Initial cells density: 5 x E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Other: The flasks were placed on a shaking incubator (Ito Seisakusho Ltd, Tokyo, Japan)

GROWTH MEDIUM
- Standard medium used: yes, Gorham´s medium
- Detailed composition (mg/L): NaNO3, 99.2; K2HPO4, 7.8; MgSO4-7H2O, 15.0; CaCl2-2H2O, 7.2; Na2CO3, 4.0; Na2-CO3SiO3 -9H2O, 11.6; EDTA, 1.0; citric acid, 1.2;
and ferric citrate, 1.2; plus Gaffron trace element solution, 0.08 ml. Gaffron trace element solution consists of the following (g/L): H3BO3, 3.100; MnSO4-4H2O, 2.230; ZnSO4-7H2O, 0.287; (NH4)6Mo7O24-4H2O, 0.088; CuSO4-5H2O, 0.125; Co(NO3)2-6H2O, 0.146; Al2(SO4)3K2SO4-24H2O, 0.474; NiSO4(NH4)2SO4-6H2O, 0.198; Cd(NO3)2-4H2O, 0 .154; Cr(NO3)3-7 H2O, 0.037; V2O4(SO4)3-16H2O, 0.035; Na2WO4-2H2O, 0.033; KBr, 0.119; and KI, 0.083

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous light
- Light intensity and quality: 4000 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: after 0, 24, 48, 72, and 96 h by electronic particle counter (Coulter counter TA-II, Coulter Electronics, Hialeah, FL, USA)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5 - 2.0
- Range finding study: yes
Reference substance (positive control):
not specified
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
70 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: S. capricornutum
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
170 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: C. vulgaris

Description of key information

ErC50 (96 h): 70.0 mg/L (S. capricornutum, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
70 mg/L

Additional information

There are three scientific publications available investigating the toxicity of the substance to different species of aquatic freshwater green algae. All studies are performed under non-GLP conditions but according to internationally accepted guidelines. It is considered to be justified to use the available studies in a weight of evidence approach to assess the toxicity to aquatic algae. The reported ErC50 values were 70.0 mg/L (96 h, S. capricornutum), 170.0 mg/L (96 h, C. vulgaris), and 85 mg/L (48 h, S. subspicatus/D. subspicatus).