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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:
other: study conducted in GLP accreditated laboratory according to OECD 201 and EC C.3
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples for analysis of concentrations of dissolved tin, total tin and methanesulfonate in the test medium were taken at test start and at test termination.
Vehicle:
no
Details on test solutions:
A stock solution with 10 mg test item in 1 L growth medium was prepared in a sterile glass bottle, mixed vigorously and treated in an ultrasonic water bath for 5 minutes. Aliquots were taken and diluted with growth medium to obtain the five test concentrations.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen (Germany)
- Age of inoculum (at test initiation): Exponential growth
- Method of cultivation: the stock cultures are maintained fulfilling the criteria of OECD 201. prior to testing a pre-culture is established in test medium to obtain exponentially-growing algae for the test.

ACCLIMATION
- Acclimation period: Until exponential growth was reached and acc. to OECD 201
- Culturing media and conditions (same as test or not): same as test medium (OECD medium)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol Ca+Mg/L (nominal)
Test temperature:
between 22.0°C and 22.1°C
pH:
between 7.74 and 8.36
Nominal and measured concentrations:
Nominal: 0 (control), 8.43, 27.8, 91.8, 303, 1000 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 ml conical glass flasks
- Type: covered with silicone-sponge caps
- Fill volume: 100 ml
- Initial cells density: 10,000 cells/ml
- Control end cells density:
- No. of organisms per vessel: 10,000 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH/TEST MEDIUM
- Standard medium used: yes, algal growth medium acc. to OECD 201 (FeCl3 * 6 H2O content slightly higher than recommended, 0.08 instead of 0.06 mg/L)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continous
- Light intensity and quality: 8000 lux (= 120 µE/m²s) ± 20 % prior to test and during test

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (CASY 1 Model TT, Schärfe System, Germany)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.3
- Range finding study: yes
- Test concentrations: 0.05, 0.5 and 5 mg/L
- Results used to determine the conditions for the definitive study: Concentrations resulted in an inhibition of biomass of 28, 91 and 79 % compared to controls, respecitvely.
Reference substance (positive control):
yes
Remarks:
zinc sulfate, 3,5-dichlorophenol
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
14.4 other: µg Sn/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.35 other: µg Sn/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (total fraction)
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
9.4 other: µg Sn/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.24 other: µg Sn/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 179 other: µg Sn/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Results with reference substance (positive control):
Zinc sulphate proved inhibitory at expected concentrations.
Reported statistics and error estimates:
NOEC and LOEC values were analysed by Williams t-test.

Analysis was performed for total tin and dissolved tin and at the low concentrations tested, a high recovery of tin was obtained for all replicates for the start of exposure. The levels of tin fell during the study, perhaps adsorbing into organic matter.

Validity criteria fulfilled:
yes
Conclusions:
In this algal growth inhibition test according to OECD guideline 201, an inhibitory effect of tin (Sn) in the form of tin(II)methansulfonate on the growth of exponentially growing Pseudokircheriella subspicata could be observed after 72 h exposure. For the effect on biomass, a 72 h EC10 of 0.35 µg/L and 0.24 µg/L (measured) was determined for total tin and dissolved tin, respectively. Based on the growth rate of P. subcapitata, the 72 h EC10 for total tin was 14.4 µg/L (measured) and the 72 h EC10 for dissolved tin was 9.4 µg/L (measured). Further, a 72 h EC50 of > 179 µg dissolved tin/L (measured) was determined. At this concentration 21% inhibition of algae growth rate was observed.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other:
Remarks:
validity not assessable, exposure concentration chemically not verified, results and methods not well reported
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Vehicle:
yes
Details on test solutions:
The test substance was dissolved in acetone to make a stock solution of 10000 mg Sn/L.
Test organisms (species):
other: Scenedesmus quadricauda, Ankistrodesmus falcatus var. acicularis, Anabaena flos-aquae and Lake Ontario algae
Details on test organisms:
Three axenic algal cultures were used: Scenedesmus quadricauda (culture collection No. 11, Dr P. Healey, Freshwater Institute, Winnipeg, Man.), Ankistrodesmus falcatus var. acicularis and Anabaena flos-aquae (Ontario Ministry of Natural Resources, P.O. Box 213, Rexdale, Ont.). Each was grown in 100 mL of a modified CHU-10 medium (Chu 1942) at 20°C on a rotary shaker (100 rpm) under conditions of 18 h of light (5000 lx) and 6 h of darkness. No chelator (ethylenediaminetetraacetic acid) was added to the medium to avoid a high degree of chelation of the metals. Only the major nutrients, nitrate and phosphate, and some bicarbonate to buffer the medium to pH 8, were present. When the cells reached the logarithmic phase of growth (about 1 week), they were used as inoculum.
Toxicity of tin chloride on a natural algal community was tested with water from a sampling site in Lake Ontario near Hamilton, Ont..
Test type:
static
Water media type:
freshwater
Limit test:
no
Remarks on exposure duration:
Primary productivity experiment: 4 hours; Algal reproduction experiment: 8 days
Test temperature:
20°C
pH:
8
Nominal and measured concentrations:
0 - 50 mg as Sn(II) 2+/L
Details on test conditions:
PRIMARY PRODUCTIVITY EXPERIMENTS
Primary productivity was measured by the amount of 14C-carbonate taken up by algae over a 4-h period. One militre of the culture (approx. 7 x 10^5 cells/mL) was added to 13.9 mL of CHU-10 medium containing various tin compounds at 0 - 50 mg as tin/L in a 25-mL Erlenmeyer flask. After a 24-h incubation at 20°C under conditions described above, a 0.1-mL aliquot of 7.4 x 10^4 Bq/mL 14C-sodium carbonate (Amersham/Searle, 2.2 x 10^9 Bq/mmol) was added to each flask. The flasks were tightly capped with rubber stoppers wrapped in aluminum foil. A similar set of flasks was incubated in the dark. After further 4-h incubation, the cells were fixed with 0.05 mL of neutralized formalin. They were filtered through a 0.45-µm mebrane filter and rinsed rapidly with 10 mL fresh CHU-10 medium to remve extracellular 14C-sodium carbonate. Filters containing radioactive labeled cells were dissolved in 10 mL PCS scintillation counting fluor (Amersham/Searle). Radioactivity was measured by liquid scintillation counter with an automatic quenching factor and a 10000 dpm upper limit. Radioactivity taken up by algae in the dark (<5% of total) was subtracted from the total radioactive counts. Radioactivity in the algae not exposed to tin compounds (control) was taken as 100%. The concentration causing a 50% reduction in primary productivity (median inhibition concentration, IC50) was estimated.
Toxicity of tin chloride on a natural algal community was tested with water from a sampling site in Lake Ontario near Hamilton, Ont. To compensate for the smaller number of algae, the volume of water was increased from 15 to 100 mL. One hundred and fifty microlitres of 3.7 x 10^5 bq/mL 14C-sodium carbonate was used. Other conditions were the same as in culture experiments.

ALGAL REPRODUCTION EXPERIMENT
The effects of tin on reproduction (growth) of A. falcatus were measured spectrophotometrically witha Klett-Summerson Photoelectric colorimeter. One millitre of the inoculum was added to 49 mL CHU-10 medium with and without tin compounds in 300-mL Nephelo culture flask with side arm (Bellco Co., Vineland, NJ). At various intervals during incubation, the growth medium was tilted into the side arm which was then inserted into the colorimeter (with red filter No. 66). Because no medium is withdrawn from the flask for determination, this technique was more convenient and less prone to contamination. klett units were converted to cell number using a standard curve relating klett units with cell number.
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
> 10 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: growth inhibition
Remarks on result:
other: Based on data of A. falcatus and based on visual estimation
Duration:
4 h
Dose descriptor:
IC50
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: growth inhibition
Remarks on result:
other: Based on data of A. falcatus
Duration:
4 h
Dose descriptor:
IC50
Effect conc.:
> 5 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: growth inhibition
Remarks on result:
other: Based on data of A. flos-aquae
Duration:
4 h
Dose descriptor:
IC50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: growth inhibition
Remarks on result:
other: Based on data of S. quadricauda.
Duration:
4 h
Dose descriptor:
IC50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: growth inhibition
Remarks on result:
other: Based on data of Lake Ontario algae
Details on results:
EFFECTS ON SCENEDESMUS QUADRICAUDA
For S. quadricauda, inorganic tin compounds were relatively nontoxic. Tin (II) had no effect on algal primary production even at levels as high as 50 mg/L.


Validity criteria fulfilled:
not specified
Conclusions:
The inhibition of growth of several freshwater algae species by exposure to tin (Sn) in the form of tin chloride (SnCl2) was assessed in this 4 h primary productivity test, and an 8 d algae reproduction (growth) test in the style of the OECD guideline 201. The following effect values were determined for the different algae species: IC50 (4h, Ankistrodesmus falcatus): 14 mg/L (nominal concentration of Sn(II) 2+); IC50 (4h, Anabaena flos-aquae): > 5 mg/L (nominal concentration of Sn(II)2+); IC50 (4h, Scenedesmus quadricauda): > 50 mg/L (nominal concentration of Sn(II)2+); IC50 (4h, Lake Ontario algae): > 50 mg/L (nominal concentration of Sn(II)2+) and NOEC (8 d, A. falcatus): > 10 mg/L (nominal concentration of Sn(II)2+).

Description of key information

Sn has a moderate acute toxic potential and a high chronic toxic potential to freshwater algae, as indicated by a 72 h EC50 of > 179 µg dissolved tin/L (measured, growth rate), and a 72 h EC10 for dissolved tin of 9.4 µg/L (measured, growth rate).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
9.4 µg/L

Additional information

One reliable (RL1), GLP confirm algae toxicity study for Sn was performed with tin(II)methanesulfonate. In a worst-case approach, all observed toxicity is attributed to Sn:

 

In this algal growth inhibition test, according to OECD guideline 201, an inhibitory effect of tin (Sn) in the form of tin(II)methansulfonate on the growth of exponentially growing Pseudokircheriella subspicata could be observed after 72 h exposure. For the effect on biomass, a 72 h EC10 of 0.35 µg/L and 0.24 µg/L (measured) was determined for total tin and dissolved tin, respectively. Based on the growth rate of P. subcapitata, the 72 h EC10 for total tin was 14.4 µg/L (measured) and the 72 h EC10 for dissolved tin was 9.4 µg/L (measured). Further, a 72 h EC50 of > 179 µg dissolved tin/L (measured) was determined (Wenzel et al. 2006). At this concentration 21% inhibition of algae growth rate was observed.

 

In a supporting study of limited reliability (RL3), inhibition of growth of several freshwater algae species by exposure to tin (Sn) in the form of tin chloride (SnCl2) was assessed in this 4 h primary productivity test, in the style of the OECD guideline 201. The following effect values were determined for the different algae species and indicate a low toxicity potential of Sn to algae: IC50 (4h, Ankistrodesmus falcatus): 14 mg/L (nominal concentration of Sn(II) 2+); IC50 (4h, Anabaena flos-aquae): > 5 mg/L (nominal concentration of Sn(II)2+); IC50 (4h, Scenedesmus quadricauda): > 50 mg/L (nominal concentration of Sn(II)2+); IC50 (4h, Lake Ontario algae): > 50 mg/L (nominal concentration of Sn(II)2+), and NOEC (8 d, A. falcatus): > 10 mg/L (nominal concentration of Sn(II)2+).

 

Data on marine organisms does not exist.

 

Based on the reliable, GLP confirm algae toxicity study it is concluded that Sn has a low acute toxic potential and a high chronic toxic potential to freshwater algae, as indicated by 21% growth inhibition of algae at 179 µg dissolved tin/L (measured), and a 72 h EC10 for dissolved tin of 9.4 µg/L (measured, growth rate).

 

Read across justification:

Tin difluoride is an inorganic solid at room temperature and consists of the tin cation and fluoride anions. Based on the solubility of tin difluoride in water (300-428 g/L according to handbook data (Merck, 2006; Gestis, 2015)), a complete dissociation of tin difluoride resulting in tin and fluoride ions may be assumed under environmental conditions. The respective dissociation is reversible and the ratio of the salt /dissociated ions is dependent on the metal-ligand dissociation constant of the salt, the composition of the solution and its pH. The metal-ligand equilibrium constant for the formation of tin difluoride is reported as follows (Japan Nuclear Cycle Development Institute, 1999):

 

Sn2++ 2F-SnF20(log K =7.74)

 

Thus, it may reasonably be assumed that based on the tin-difluoride formation constant, the respective behaviour of the dissociated tin cations and fluoride anions in the environment determine the fate of tin difluoride upon dissolution with regard to (bio)degradation, bioaccumulation, partitioning resulting in a different relative distribution in environmental compartments (water, air, sediment and soil) and subsequently determine its ecotoxicological potential.

Therefore, in the assessment of the ecotoxicity of tin difluoride, read-across to data for fluoride and soluble tin substances is applied since only the ions of tin difluoride are available in an aqueous environment and determine the environmental fate and toxicity. Read-across to environmental fate and toxicity studies of soluble tin salts, including tin dichloride and tin methane sulfonic acid, is therefore appropriate and scientifically justified.