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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study conducted under GLP. However, the presence of a precipitate in the test solution (most likely insoluble tin oxide) may have caused some physical toxicity in the test system and analytical results indicated a loss of the test material over the course of the study.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
and EU Directive 92/69/EEC, Method C.3.
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the test substance. Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to the addition of algae. At test termination, samples were collected from the pooled replicates of each treatment and control group. All samples were collected in glass vessels and were analyzed immediately without storage. The 15 mg/L stock was also sampled at test initiation.
- Sample storage conditions before analysis: analyzed immediately without storage.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A primary stock solution was prepared by dissolving Tin (II) 2-Ethylhexanoate in fresh water algal medium at a nominal concentration of 15 mg/L. The test material was weighed on a small piece of Teflon which was added directly to media. The stock was sonicated approximately 30 minutes and stirred approximately 20 hours using a magnetic stir plate and Teflon coated stir bar and appeared slightly cloudy. After mixing the stock appeared slightly cloudy. A secondary stock was prepared by diluting 400 mL of the 15 mg/L stock solution to achieve the highest nominal test concentration of 6.0 mg/L. The secondary stock was proportionally diluted with freshwater algal medium to prepare the four additional test solutions at nominal concentrations of 0.25, 0.55, 1.2 and 2.7 mg/L. Test concentrations were not corrected for percent active ingredient in the test substance.
- Eluate: No data
- Differential loading: No data
- Controls: Negative (culture medium) control under static conditions for 72 hours. Six replicate test chambers were in the contol group.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All test solutions appeared clear and colorless. At test termination test solutions less than or equal to 1.2 mg/L had no visible particulates or surface slicks, however, the 2.7 and 6.0 mg/L test solutions had white precipitation.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Freshwater green algae
- Strain: Pseudokirchneriella subcapitata (UTCC #37)
- Source (laboratory, culture collection): University of Toronto Culture Collection (UTCC) and were maintained in culture medium at Wildlife International, Ltd Easton, Maryland
- Age of inoculum (at test initiation): Cultures has been actively growing in culture medium for at least 2 weeks prior to test initiation, and had been transferred to fresh medium three days prior to test initiation.
- Method of cultivation: Cultures has been actively growing in culture medium for at least 2 weeks prior to test initiation, and had been transferred to fresh medium three days prior to test initiation.

ACCLIMATION
- Acclimation period: Approximately 2 weeks
- Culturing media and conditions (same as test or not): Same
- Any deformed or abnormal cells observed: No data
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
day 0- measurement 1 = 24.0 deg C measurement 2 = 25.2 deg C
1 1 = 25.3 2 = 25 .2
2 1 = 25.3 2 = 25.1
3 1 = 25.2 2 = 25.1
pH:
7.8 to 10.1
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Nominal concentrations were: Negative control, 0.25, 0.55, 1.2, 2.7 and 6.0 mg/L
Measured concentrations were: 0.24, 0.54, 1.2, 2.7, 6.1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 -mL Erlenmeyer flasks plugged with foam stoppers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 ml
- Aeration: Test flasks were shaken continously at 100 rpm
- Initial cells density: Approximately 10,000 cells/mL
- No. of organisms per vessel: Approximately 10,000 cells/mL
- No. of vessels per concentration (replicates): Three
- No. of vessels per negative control: Six
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM

- Standard medium used: Yes, the algal cells were cultured and tested in freshwater algal medium. Stock nutrient solutions were prepared by adding reagent- grade chemicals to purified Wildlife Internatioal, Ltd. well water (NANOpure water). Test medium then was prepared by addiing appropiate volumes of the stock solutions to purified well water (NANOpure water).


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory well water
- Culture medium different from test medium: no
- Intervals of water quality measurement: performed in December 2005

OTHER TEST CONDITIONS
- Adjustment of pH: yes to 8.0 using 10% HCL and then sterilized (0.22 um) prior to use.
- Photoperiod: Continous
- Light intensity and quality: The algae were held under continous cool-white fluorescent lighting throughout the test. The target light intensity was 6000 +/- 20% lux. Light intensity was measured at five locations surrounding the test flasks on the shaker table at test initation using a SPER Scientific Model 840006C light meter.
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Area under the curve (biomass) and growth rate
- Determination of cell concentrations: Cell counts were performed using a hemacytometer and microcsope
- Chlorophyll measurement: No data


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Guideline
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Nominal test concentrations were selected in consultation with the Sponsor and were based upon results of exploratory range finding tests.
- Test concentrations: negative control, 0.25, 0.55, 1.2, 2.7 and 6.0 mg/L
- Results used to determine the conditions for the definitive study: No data
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.9 mg/L
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 6.2 and 7.7 mg/L. The ErC50 value was above the concentrations tested and should be considered an extrapolated value.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.54 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.22 mg/L
Basis for effect:
biomass
Remarks on result:
other: Based on the calculated EC10
Details on results:
Inhibition of area under the growth curve in the 0.24, 0.54, 1.2, 2.7 and 6.1 mg/L treatment groups was 13, 20, 61, 79 and 92%, respectively, relative to the negative control. Treatment related effects were apparent in all test concentrations.

Inhibition of growth rate in the 0.24, 0.54, 1.2, 2.7 and 6.1 mg/L treatment groups was 1.1, 3.6, 18, 29 and 46%, respectively, relative to the negative control. Treatment related effects were apparent in the three highest test concentrations.

- Exponential growth in the control (for algal test): yes; Changes in mean cell density in the negative control replicates over the 72-hour exposure period indicated that exponential growth of cells occurred in those replicates
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At test  termination, a white precipitate was observed in the 2.7 and 6.0 mg/L  test solutions. Samples collected on Day 0 had recoveries of 97, 98, 102, 101 and 102% of nominal concentrations, respectively. Samples collected on Day 3 had recoveries of
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The calculations of areas under the growth curve, growth rates and percent inhibition values as well as all statistical analyses, were conducted using “The SAS System for Windows”, Version 8.02 (4).

Areas under the growth curve (biomass) and growth rates were analyzed statistically, when possible, by non-linear regression (5) to estimate EC50 values (i.e., the theoretical test concentrations that would produce a 50% reduction in biomass (EbC50) and growth rate (ErC50) for each 24-hour exposure
interval) and the corresponding 95% confidence limits. The 72-hour EC10 and EC20 values for biomass and growth rate were also determined using non-linear regression or linear interpolation. The 72-hour data for growth rate and biomass were evaluated for normality and homogeneity of variance (p=0.05) using the Shapiro-Wilk’s and Levene’s tests, respectively. The treatment groups then were compared to the negative control using Dunnett’s test (p=0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern of growth inhibition, were used to determine the NOEC relative to each parameter at 72 hours.

Biomass (area under the growth curves):
Percent inhibition (relative to control) at 0-72 hours, by Day 0 measured  test concentration:
0.24 mg/L: 13%*
0.54 mg/L: 20%*
1.2 mg/L: 61%*
2.7 mg/L: 79%*
6.1 mg/L: 92%*
* Statistically significantly different (p <0.05) from the negative  control 

72-h Effects Values (95% confidence limits):
NOEC(b)  0.22 mg/L (based on calculated EbC10)
EbC10     0.22 (0.14-0.33) mg/L**
EbC20     0.37 (0.26-0.52) mg/L
EbC50     1.0 (0.83-1.3) mg/L
** Value should be considered an extrapolated value
NOEC = estimated No Observed Effect Concentration.

Growth Rate:
Mean growth rate (percent inhibition relative to control) at 0-72 hours,  by Day 0 measured test concentration:
Negative control: 0.087 cells/mL (N/A)
0.24 mg/L: 0.086 cells/mL (1.1%)
0.54 mg/L: 0.084 cells/mL (3.6%)
1.2 mg/L: 0.071 cells/mL (18%)*
2.7 mg/L: 0.061 cells/mL (29%)*
6.1 mg/L: 0.046 cells/mL (46%)*
* Statistically significantly different (p <0.05) from the negative  control

72- h Effects Values (95% confidence limits):
NOEC(r)  0.54 mg/L
ErC10     0.77 (0.57-0.96) mg/L
ErC20     1.6 (1.3-1.9) mg/L
ErC50     6.9 (6.2-7.7) mg/L**
** Value should be considered an extrapolated value
NOEC = estimated No Observed Effect Concentration.

Validity criteria fulfilled:
yes
Conclusions:
The freshwater green alga, Pseudokirchneriella subcapitata, was exposed for 72 hours to five test concentrations of Tin bis(2-ethylhexanoate) ranging from 0.24 to 6.1 mg/l. The 72-hour EbC50 value, based on area under the growth curve (biomass), was 1.0 mg/L, with a 95% confidence interval of 0.83 and 1.3 mg/L/ The 72-hour ErC50 value, based on growth rate, was 6.9 mg/L, with 95% confidence interval of 6.2 and 7.7 mg/L. The ErC50 value was above the concentrations tested and should be considered an extrapolated value. The 72-hour NOEC for the most sensitive endpoint (biomass) was based on the calculated EC10 of 0.22 mg/L.
Executive summary:

The objective of this study was to determine the toxicity of Tin bis(2 -ethylhexanoate) to the freshwater green algae, Pseudokirchneriella subcapitata, during a 72 hour exposure period.

The algae was exposed to a geometeric series of five test concentrations, and a negative (culture medium) control under static conditions for 72 hours. Three replicate test chambers were maintained in each treatment group with six replicate test chambers in the control group. Nominal test concentrations were selected in consultation with the Sponsor and were based upon results of exploratory range finding toxocity tests. Nominal test concentrations were 0.25, 0.55, 1.2, 2.7 and 6.0 mg Tin (II) 2 -Ethylhexanoate/L (mg/L). Measured test concentrations were determined from samples of test medium collected from each treatment and control group at the beginning and end of the test.

At test initiation an inoculum of Pseudokirchneriella cells was added to each chamber to achieve a nominal concentration of approximately 10,000 cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities, which were subsequently used to calculate areas under the growth curve (biomass) and growth rates. Areas under the growth curve and growth rates were used to calculate percent inhibition values relative to the control over the 72 -hour exposure period. EbC50 and ErC50 values were calculated, when possible, based upon biomass and growth rate, respectively, for each 24 -hour interval of the exposure period. The 72 -hour EC10 and EC20 values also were calculated for biomass and growth rate. The no-observed-effect-concnetration (NOEC) was determined relative to biomass and growth rate at 72 hours through evaluation of the statistical results and the concentration-response pattern of growth inhibition.

Description of key information

The 72-hour NOEC and ErC50 for Pseudokirchneriella subcapitata exposed to tin bis(2-ethylhexanoate) was 0.54 and 6.9 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
6.9 mg/L
EC10 or NOEC for freshwater algae:
0.54 mg/L

Additional information

In a valid toxicity study with Pseudokirchneriella subcapitata (Klimisch score = 2), algal cells were exposed to nominal test concentrations of 0, 0.25, 0.55, 1.2, 2.7 and 6.0 mg/L tin bis(2-ethylhexanoate). Measured test concentrations were determined from samples of test medium collected from each treatment and control group at the beginning and end of the test. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities. Areas under the growth curve and growth rates were used to calculate percent inhibition values relative to the control over the 72 -hour exposure period. The 72-hour NOEC and EC50 based on growth rate were 0.54 and 6.9 mg/L, respectively. The 72-hour NOEC and EC50 based on biomass were 0.22 and 1.0 mg/L, respectively. Since the use of the average specific growth rate for estimating toxicity is scientifically preferred (OECD 201), this toxicity value was selected as key for this endpoint.