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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. Exposure concentrations, measured on the basis of total Sn, were converted to and reported as the named substance [MMT(2-EHMA)] using the conversion factor [743.7/118.71] consisting of the Mol weights of the test substance and Sn, respectively. All measured Sn was attributed to the named substance. This approach includes more water-soluble tin impurities and hydrolysis products. The preparation of the WSF allowed both a solubilized portion of the test substance and the hydrolysis products to be present in the study. Analytical repeatability was low due to the low solubility of the named substance. This is not unexpected given the low water solubility of the named substance and its instability in water. At all nominal concentrations <9.9 % of the WSF, the measured Sn concentrations were less than the analytical level of detection (LOD). The measured Sn concentrations showed a linear relationship with the percentage of the dilutions of the WSF (plotted on a Ln scale), allowing for an extrapolation of Sn concentrations where measured values were
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The title of the report was changed in "Determination of the effect on the ..."; the glass fiber filter used for filtering the test substance water soluble fraction (WSF) was Whatman GF/C 47 mm; At the start and at the end duplicate samples for analysis w
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test substance concentrations
- Sampling method: 100 mL samples were taken
- Sample storage conditions before analysis: refrigerated
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to its low water solubility, a water solube fraction (WSF) of the test substance was prepared. An amount of test substance was added directly to an appropriate volume of algal medium in a one litre flask, followed by stirring for 16-20 hours using a magnetic stirrer. Following stirring, the medium was allowed to stand for a few hours to enable phase separation. Then the dispersion was filtered, passing a Whatman GF/C glass fiber filter (47 mm). The first 10-50 mL filtrate was discarded and the last 10 mL, containing remaining undissolved test substance, was not filtered. The test solution was diluted with algal medium to prepare a next lower test solution and so on until a dilution series had been prepared.
- Eluate: algal medium
- Differential loading: 0.1044 g in one liter of algal medium
- Evidence of undissolved material (e.g. precipitate, surface film, etc): prepared WSF contained white solid particles prior to filtering.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Scenedesmus subspicatus
- Source (laboratory, culture collection): Collection of Algal Cultures, Institute for Plant Physiology, University of Göttingen, Göttingen, Germany
- Age of inoculum (at test initiation): No data
- Method of cultivation: A pre-culture of algae in the exponential growth phase was prepared as detailed in OECD guideline no. 201.
- Initial cell concentration: 0.3 x 10E4 cells/mL


ACCLIMATION
- Acclimation period: no data
- Culturing media and conditions (same as test or not): A pre-culture was diluted in algal medium, same as test
- Any deformed or abnormal cells observed: no data
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
24.2 mg equivalent CaCO3/L
Test temperature:
21.4 - 23.3°C (mean 22.8 °C)
pH:
7.9 - 8.0
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Nominal concentrations: 0, 0.003, 0.01, 0.03, 0.3, 9.9, 33 and 99 % of a WSF
Geometric mean concentrations: 0.007, 0.023, 0.072, 0.76, 28, 95 and 294 micrograms Sn/L effect values were calculated on the basis of the geometric mean of the measured and extrapolated Sn concentrations at the start and end of the test.
Details on test conditions:
TEST SYSTEM
- Test vessel: Flask
- Type (delete if not applicable): open / closed- No data
- Material, size, headspace, fill volume: One liter glass flask
- Aeration: NA
- Type of flow-through (e.g. peristaltic or proportional diluter): NA
- Renewal rate of test solution (frequency/flow rate): NA
- Initial cells density: 3 X 10^4 cells.ml-1
- Control end cells density: 3 X 10^4 cells.ml-1
- No. of organisms per vessel: No data
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: NA

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Ultra pure water
- Total organic carbon: No data
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/mg ratio: No data
- Conductivity: No data
- Culture medium different from test medium: No
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: No data
- Adjustment of pH: No, at the start of the test the pH of the algal medium (control and highest test substance concentration) was found to be 7.9-8.0. At the end of the test, in absence and presence of algal cells, the pH was found to have remained stable during the test (pH 7.8 – 8.3).
- Photoperiod: No data
- Light intensity and quality: Light intensity radiated by the fluorescent lamps was within the standard range of 60-120 µmol.s-1.m-2
- Salinity (for marine algae): NA

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement: No data
- Other: No data

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentration than requested by guideline: NA
- Range finding study
- Test concentrations: 0, 0.01, 0.10, 1.0, 9.9 and 99 % of the WSF of MMT (EHMA)
- Results used to determine the conditions for the definitive study: The range-finding test revealed an expected NOEC of approximately 0.01 % of the WSF. On the basis of the results of the range-finding test, a test dilution series of 0, 0.003, 0.01, 0.03, 0.3, 10 and 33 % WSF of MMT (EHMA) was proposed and approved by the sponsor. As the measured Sn concentration at different tests was not consistent, it was decided to repeat the test and to add 100 % WSF to the concentration series. The chemical analysis of Sn of MMT (EHMA) resulted in 170 µg Sn.1-1 . (100 % WSF t = 0), and 180 µg Sn.1-1(100 % WSF t = 3 d), respectively. The test substance Sn concentration was stable during the test.

METHOD OF CALCULATION:
- Substance concentration: The named substance concentration was calculated from the measured tin (Sn) concentrations using the conversion factor [743.7/118.71] consisting of the Mol weights of the substance and Sn, respectively. All measured Sn was fully attributed to the named substance, MMT(2-EHMA).
- ECr values: DEBtox software, according to the Dynamic Energy Budgets Theory developed by Kooijman and Bedaux (1981), was used to calculate the effect concentration (EC) values. A Parametric model developed by Kooijman et.al (1981) was used to calculate the EC values with respect to the growth rate during exponential growth (ECr), assuming a constant error per measurement.
- ECb values: The EC values with respect to area under the growth curve were calculated by linear interpolation of a plot of the percentage reduction in growth against the log concentration of the test substance.
- Growth rate: The average specific growth rate was determined according to the OECD guideline.
- NOEC: The NOEC was determined as the highest concentration at which no (statistically) significant inhibition of the average specific growth rate was observed.
- NEC: Where possible, the NEC was calculated using the DEBtox software package according to the Dynamic Energy Budgets Theory developed by Kooijman and Bedaux (1981). Model parameters for population growth and their asymptotic standard deviation and correlation coefficients were estimated. The NEC was calculated from the Profile Ln Likelihood function. This method offers a completely different basis for calculating No Effect values and offers complementary information to the traditional statistical NOEC or EC10/20 approach.

STABILITY OF THE TEST CHEMICAL SOLUTIONS:
The test substance has a low water solubility and is unstable in water. Based on the analytical method used (i.e., analysis of total Sn) it is not possible to conclude if the test substance was stable during the test.

OTHER:
A range-finding test was performed as a simplified version of the OECD guideline 201. The nominal test substance concentration series were 0, 0.01, 0.10, 1.0, 9.9 and 99 % of the WSF of the substance. The test was carried out in duplicate with four controls containing algae only and a single background series containing test substance without algae. Other test conditions were similar to the growth inhibition test as described above. After 3 days of incubation, algal densities and bio-volume were determined. The morphology of the algae was examined visually with the aid of a microscope at the start and end of the test. Test substance concentrations in the range-finding study were monitored using total Sn analysis, as mentioned previously.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7 and/or 3,5-dichlorophenol on an approximately yearly basis.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.81 mg/L
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.8 mg/L
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.6 mg/L
Basis for effect:
growth rate
Details on results:
- Observation of abnormalities (for algal test): No
- Unusual cell shape: Microscopic inspection of the morphology of algal cells in the pre-culture at the start of the test revealed normal cells. At the end of the test no abnormal cells were observed in the cultures containing different concentrations of MMT EHMA).
- Color differences: None
- Flocculation: None
- Adherence to test vessels: No data
- Aggregation of algal cells: No data
- Other: No data
- Any stimulation of growth found in any treatment: The OECD Guideline No. 201 recognises two validity criteria, i.e. a sufficient control growth rate, and a limited increase of the test medium pH value during the test (one pH unit). The control growth rate (0.056 h-1) is higher than the minimal cell multiplication factor of 16 during a three day test given in the Guideline (corresponding to a growth rate of 0.038 h-1). The maximum pH increase in the control cultures was 0.3, which is well below the limit given in the Guideline. The test therefore met the validity criteria of the Guideline.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: For the final test the WSF was prepared by a test substance loading of 0.1044 g in one liter of algal medium, giving a clear suspension containing white solid particles. The filtrated clear solution was diluted with algal medium to prepare a lower test dilution. That test solution was diluted with algal medium to prepare a next lower test solution and so on until a dilution series had been prepared.
Results with reference substance (positive control):
REFERENCE SUBSTANCE: The sensitivity of the test system is checked with K2Cr2O7 and/or 3,5-dichlorophenol on an approximately yearly basis. The results demonstrate a constant quality of the test system during an extended period of at least 15 years. The resulting EC50 values are also comparable to the mean values found in international ring tests.
Reported statistics and error estimates:
STATISTICAL METHODS: Statistical significance of the growth inhibition was determined with a one-tailed t-test (significance level = 5 %).
It was concluded that the ErC50 of >1.8 mg/L as MMT(2-EHMA), based on the geometric mean of the measured concentrations of Sn, is the best estimate of the mean toxicity of the test substance to the fresh water alga Scenedesmus subspicatus. A calculated NEC was not possible. The NOEC was determined to be 0.6 mg/L as MMT(2-EHMA) (95 micrograms/L as Sn).
RESULTS: RANGE FINDING
- Measured substance concentrations: The chemical analysis of the substance (as Sn) in a 100% Water Soluble Solution (WSF) resulted in 170 micrograms Sn/L on t=0 and 180 micrograms Sn/L on t=3 days), respectively. The Sn concentration was thus stable during the range-finding test.
- Results: The range-finding test revealed an expected NOEC of approximately 0.01% of the WSF. Based on the results of the range-finding test, it was decided to test 0, 0.003, 0.01, 0.03, 0.3, 10 and 33% WSF. However, as the measured Sn concentration at different levels was not consistent, it was decided to repeat the (main) test and add 99% WSF to the concentration series.
RESULTS: EXPOSED
- Nominal/measured concentrations: Nominal concentrations were 0, 0.003, 0.01, 0.03, 0.3, 10, 33 and 99% of the WSF. At all nominal concentrations <9.9% WSF, the measured Sn concentrations were less than the analytical level of detection (LOD). The mean recovery of the Sn concentration at the end of the test was 89% and supports the conclusion of the range finding test that the test substance was stable during the test period. The measured Sn concentrations showed a linear relationship with the percentage of the dilutions of the WSF (plotted on a Ln scale), allowing for an extrapolation of Sn concentrations where measured values were <LOD. Therefore, the effect values were calculated on the basis of the geometric mean of the measured and extrapolated Sn concentrations at the start and end of the test. The geometric mean measured concentrations corresponding to the nominal test substance concentrations of 0.003, 0.01, 0.03, 0.3, 10, 33 and 99% WSF were 0.007, 0.023, 0.072, 0.76, 28, 95 and 294 micrograms Sn/L, respectively.
Remark: There was a difference in the measured Sn concentration of the WSFs between the range-finding and the final growth inhibition tests. Attempts to obtain consistent Sn concentrations in WSFs prepared at different times with the same method did not succeed. Even the addition of energy with the aid of an Ultra Turrax homogenizer did not yield a higher Sn concentration.
Some algal density determinations deviated from the expected range. The reason for this variation is uncertain, but might be due to the tendency of the test organism for a synchronic growth pattern. Nevertheless, no determinations were regarded as outliers, as the averages revealed a normal pattern.
- Effect values (micrograms Sn/L and micrograms MMT(2-EHMA)/L respectively, both with 95% confidence limits):
micrograms Sn/L micrograms MMT(2-EHMA)/L
NOEC 95 595
ErC10 130 814
ErC50 >294 >1840
ErC90 >294 >1840
EbC10 4.8 (0.76-28)* 30 (4.8-173)*
EbC50 >294 >1840
EbC90 >294 >1840

* = range between tested concentrations
NOEC = estimated No Observed Effect Concentration
ErC values = Effect Concentration with regard to the growth rate
EbC values = Effect Concentration with regard to area under the growth curves
RESULTS CONTROL: Microscopic inspection of the morphology of algal cells in the pre-culture at the start of the test revealed normal cells. At the end of the test, no abnormal cells were observed in the cultures containing different concentrations of the test substance.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditons of the test, the ErC50 of MMT(2-EHMA) was estimated to be >1.8 mg/L.
Executive summary:

The toxicity of MMT(EHMA) to the fresh water green alga Scendesmus subspicatus was determined in a 72 -h growth inhibition tests following the OECD testing guideline 201. A saturated solution of the test substance (Water Soluble Fraction) was prepared with algal growth medium. Repeated dilutions of the WSF resulted in a testing concentration series of 0, 0.003, 0.01, 0.03, 0.3, 9.9, 33 and 99 % of the WSF of MMT(EHMA) in algal growth medium. It was concluded that the ErC50 value >1840 µg MMT(EHMA)/l, based on the geometric mean of the measured tin concentrations is the best estimate of the mean effect concentration of MMT(EHMA) to the fresh water algae Scendesmus subspicatus. The NOEC value in the S. subspicatus test system was 95 µg as tin/L, which is equivalent to 595 µg/L.

Description of key information

Toxicity to algae: ErC50 >1840 µg/L in Scenedesmus subspicatus; NOEC 595 µg/L (OECD 201)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
0.595 mg/L

Additional information

The toxicity to the freshwater green alga Scenedesmus subspicatus was determined in a 72 hour OECD 201 study. Of several parametric models available, the model that assumes an effect on the growth rate and exponential growth (ErC50 values) appeared to fit the data. The EC50 with respect to growth rate and geometric mean (ErC50) was found to be >1840 µg/L. The average specific growth rate and percentage inhibition were determined. The highest concentration that produced no oberservable effects was 595 µg/L. However, the effects found gave no consistent concentration effect relationship.