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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 16 June 2014 and 29 August 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: BMS-587172-01
- Substance type: Chemical intermediate
- Physical state: White powder
- Analytical purity: 99.9%
- Lot/batch No.: 2G71626N
- Expiration date of the lot/batch: 27 August 2014
- Storage condition of test material:Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Stock Solution
A nominal amount of test item (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (200 µL) of this solvent stock solution was dispersed in 2 liters of culture medium with the aid of magnetic stirring for approximately 10 minutes prior to the removal of any undissolved test item by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 1.0 mg/L stock solution.
Range Finding Test
A series of dilutions was made from this stock solution to give further stock solutions of 0.10 and 0.010 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (9.0 mL) to give the required nominal test concentrations of 0.010, 0.10 and 1.0 mg/L.
Definitive Test
An aliquot (1 liter) of this stock solution was inoculated with 9.9 mL of algal suspension to give the required nominal test concentration of 1.0 mg/L.

- Eluate:
Culture Medium
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.
NaNO3 _ _ _ _ _ _ _ _ 25.5 mg/L
MgCl2.6H2O _ _ _ _ _ _ _ _ 12.16 mg/L
CaCl2.2H2O _ _ _ _ _ _ _ _ 4.41 mg/L
MgSO4.7H2O _ _ _ _ _ _ _ _ 14.6 mg/L
K2HPO4 _ _ _ _ _ _ _ _ 1.044 mg/L
NaHCO3 _ _ _ _ _ _ _ _ 15.0 mg/L
H3BO3 _ _ _ _ _ _ _ _ 0.186 mg/L
MnCl2.4H2O _ _ _ _ _ _ _ _ 0.415 mg/L
ZnCl2 _ _ _ _ _ _ _ _ 0.00327 mg/L
FeCl3.6H2O _ _ _ _ _ _ _ _ 0.160 mg/L
CoCl2.6H2O _ _ _ _ _ _ _ _ 0.00143 mg/L
Na2MoO4.2H2O _ _ _ _ _ _ _ _ 0.00726 mg/L
CuCl2.2H2O _ _ _ _ _ _ _ _ 0.000012 mg/L
Na2EDTA.2H2O _ _ _ _ _ _ _ _ 0.30 mg/L

- Controls:
The control group was maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide.
A positive control used potassium dichromate as the reference item.
- Chemical name of vehicle: dimethylformamide

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 - 105 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.098 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
> 0.098 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Results with reference substance (positive control):
Positive Control
A positive control (Harlan Study Number 41303826) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.1 mg/L; 95% confidence limits 0.91 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.51 mg/L; 95% confidence limits 0.45 – 0.59 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

Range-findingTest

The results showed no effect on growth at the test concentrations of 0.010, 0.10 and 1.0 mg/L.

 

Based on this information a single test concentration of six replicates, of 1.0 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

 

Chemical analysis of the 1.0 mg/L test preparations at 0 and 72 hours showed measured test concentrations of 0.38 and 0.25 mg/L respectively were obtained indicating that the test item was unstable over the test duration.

 

 

DefinitiveTest

Verification of Test Concentrations

Analysis of the 1.0 mg/L test preparation at 0 hours showed a measured test concentration of 0.17 mg/L. A decline in measured test concentration was observed at 72 hours to 0.055 mg/L. Given the decline in measured test concentration over the test duration it was considered appropriate to estimate the results based on the geometric mean measured test concentration in order to assume a “worst case” analysis of the data.

 

The geometric mean measured test concentration was determined to be:

 

Nominal Test Concentration
(mg/L)

Geometric Mean Measured Test Concentration (mg/L)

Expressed as a % of the 0-Hour Measured Test Concentration

1.0

0.098

56

 

Whilst the 0-hour measured test concentration obtained in the definitive test was lower than that observed in the preliminary media preparation trial and range-finding test this was considered to have had no adverse effect on the outcome of the test as no toxicity was observed at either of the concentrations obtained.

 

 

Growth Data

Cell density values determined at each sampling time and pH values at 0 and 72 hours, Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in the table below:

 

Geometric Mean Measured Test Concentration (mg/L) pH Cell Densities* (cells per mL) pH Growth Rate Yield
(cells/mL/hour) (cells/mL)
0 h 0 h 23 h 48 h 72 h 72 h 0 – 72 h % Inhibition 0 – 72 h % Inhibition
Control R1 7.6 5.14E+03 3.08E+04 2.54E+05 8.69E+05 8.4 0.072   8.64E+05  
R2 6.08E+03 1.99E+04 1.97E+05 8.91E+05 0.072   8.85E+05  
R3 5.57E+03 3.15E+04 2.79E+05 9.01E+05 0.072   8.95E+05  
R4 6.19E+03 2.03E+04 2.01E+05 8.21E+05 0.071 - 8.15E+05 -
R5 6.56E+03 2.20E+04 1.94E+05 8.16E+05 0.071   8.10E+05  
R6 6.53E+03 2.91E+04 2.36E+05 8.28E+05 0.071   8.21E+05  
Mean 6.01E+03 2.56E+04 2.27E+05 8.54E+05 0.072   8.48E+05  
SD   0.001   3.78E+04  
Solvent Control R1 7.5 5.66E+03 3.08E+04 2.12E+05 1.26E+06 8.3 0.077   1.26E+06  
R2 5.50E+03 2.74E+04 1.95E+05 1.09E+06 0.075   1.09E+06  
R3 5.74E+03 2.63E+04 2.24E+05 1.05E+06 0.074   1.04E+06  
R4 5.68E+03 2.08E+04 1.72E+05 1.16E+06 0.076 - 1.16E+06 -
R5 5.95E+03 2.65E+04 2.37E+05 1.32E+06 0.077   1.32E+06  
R6 5.60E+03 2.64E+04 2.12E+05 1.23E+06 0.076   1.22E+06  
Mean 5.69E+03 2.64E+04 2.09E+05 1.19E+06 0.076   1.18E+06  
SD   0.001   1.04E+05  
0.098 R1 7.5 5.94E+03 2.91E+04 2.60E+05 1.29E+06 8.3 0.077 [1] 1.29E+06 [9]
R2 6.27E+03 2.75E+04 1.86E+05 1.04E+06 0.074 3 1.03E+06 13
R3 5.73E+03 3.04E+04 2.83E+05 1.45E+06 0.079 [4] 1.45E+06 [23]
R4 5.36E+03 2.97E+04 2.58E+05 1.28E+06 0.077 [1] 1.28E+06 [8]
R5 5.95E+03 2.74E+04 2.18E+05 1.20E+06 0.076 0 1.19E+06 [1]
R6 6.30E+03 3.29E+04 2.81E+05 1.53E+06 0.079 [4] 1.52E+06 [29]
Mean 5.93E+03 2.95E+04 2.48E+05 1.30E+06 0.077 [1] 1.29E+06 [9]
SD   0.002   1.76E+05  

 

From the data given in the table above, it is clear that the growth rate (r) and yield (y) ofPseudokirchneriella subcapitata(CCAP 278/4) were not affected by the presence of the test item at a geometric mean measured test concentration of 0.098 mg/L over the 72-hour exposure period.

 

The test concentration of 0.098 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline.

 

Accordingly the following results were determined from the data:

 

Inhibition of Growth Rate

ErC10(0 - 72 h)           :          >0.098 mg/L

ErC20(0 - 72 h)           :          >0.098 mg/L

ErC50(0 - 72 h)           :          >0.098 mg/L

 

where ErCxis the test concentration that reduced growth rate by x%.

 

Statistical analysis of the growth rate data was carried out for the solvent control and 0.098 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences (P³0.05), between the solventcontrol and 0.098 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.098 mg/L.

 

 

Inhibition of Yield

EyC10(0 - 72 h)          :         >0.098mg/L

EyC20(0 - 72 h)          :          >0.098mg/L

EyC50(0 - 72 h)          :          >0.098mg/L

 

Where:

 

EyCxis the test concentration that reduced yield by x%.

 

Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no statistically significant differences (P³0.05), between the solvent control and 0.098 mg/L test group and therefore the NOEC based on yield was 0.098 mg/L.

 

 

Validation Criteria

The following data show that the cell concentration of the control culturesincreasedby a factor of 142 after 72 hours and the cell concentration of the solvent control cultures increased by a factor of 208 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

 

Mean cell density of control at 0 hours         :          6.01 x 103cells per mL

Mean cell density of control at 72 hours       :          8.54 x 105cells per mL

Mean cell density of solvent control at 0 hours         :          5.69 x 103cells per mL

Mean cell density of solvent control at 72 hours       :          1.19 x 106cells per mL

 

The mean coefficients of variation for section by section specific growth rate for the control and solvent control cultures were 25% and 10% respectively and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

 

The coefficient of variation for average specific growth rate for the control and solvent control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

 

 

Observationson Cultures

All test, control and solvent control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control, solvent control or test cultures.

 

 

Water Quality Criteria

The pH values of the control, solvent control and 1.0 mg/L test preparation are given in the table above. Temperature was maintained at 24 ± 1 ºC throughout the test.

 

The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 8.4 at 72 hours whilst the pH of the solvent control cultures was observed to increase from pH 7.5 at 0 hours to pH 8.3 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

 

Observations on Test Item Solubility

At the start of the test all control, solvent control and test cultures were observed to be clear colorless solutions. After the 72-hour test period all control, solvent control and test cultures were observed to be green dispersions.

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and based on the geometric mean measured test concentration gave EC50 values of greater than 0.098 mg/L. The NOEC was 0.098 mg/L.

This study showed that there were no toxic effects at the limit of solubility.
Executive summary:

A study was perford to assess the effect of the test item on the growth of the green algaPseudokirchneriellasubcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

 

 Methods……..

Information provided by the Sponsor indicated the test item had limited solubility in water. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

 

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.36 mg/L was obtained from a solvent spike solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

 

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a solution of the test item at a nominal concentration of 1.0 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solution was prepared by dispersing 200µLof a 100 mg/10 mL solvent stock solution in 2 liters of culture medium with the aid of magnetic stirring for approximately 10 minutes. After stirring any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter).

 

Due to the light sensitive nature of the test item, all test item preparation was performed under laboratory safety lighting.

 Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter.

  

Results…….

Analysis of the 1.0 mg/L test preparation at 0 hours showed a measured test concentration of 0.17 mg/L. A decline in measured test concentration was observed at 72 hours to 0.055 mg/L. Given the decline in measured test concentration over the test duration it was considered appropriate to estimate the results based on the geometric mean measured test concentration in order to assume a “worst case” analysis of the data.

Exposure ofPseudokirchneriella subcapitatato the test item gave EC50values based on the geometric mean measured test concentration of greater than 0.098 mg/L. The No Observed Effect Concentration was determined to be 0.098 mg/L.

 

This study showed that there were no toxic effects at the limit of water solubility.