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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:
Experimental start date 10 December 2018, Experimental completion date 25 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

1
Chemical structure
Reference substance name:
2-bromo-6-(4-methylbenzoyl)pyridine
EC Number:
618-079-0
Cas Number:
87848-95-1
Molecular formula:
C13H10BrNO
IUPAC Name:
2-bromo-6-(4-methylbenzoyl)pyridine
Specific details on test material used for the study:
Identification: 2-Bromo-6-(4-toluoyl)pyridine
CAS Number: 87848-95-1
Batch: 800295520
Purity: 99.5%
Physical state/Appearance: Beige powder
Expiry Date: 03 July 2020
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The test item concentration in the test samples was determined by high performance liquid chromatography with UV-VIS detection (HPLC/UV-VIS) using an external standard. The test item gave a chromatographic profile consisting of a single peak.

Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Preparation of Test Samples
The test samples were analyzed on the day of receipt. The test item was extracted from the test samples using a solid phase extraction cartridge (C18, 500 mg/ 3 mL). The cartridge waspacked with glass wool prior to being pre-conditioned with 10 mL of acetonitrile and 10 mL of water. The samples were drawn through the cartridge under reduced pressure. Subsequently, the cartridge was eluted with 5 mL of acetonitrile into a 10 mL volumetric flask. The solution was made up to the mark with water.

Storage
The samples were analyzed on the day of receipt.

Test solutions

Vehicle:
no
Details on test solutions:
Experimental Preparation
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed 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 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 50, 25, 12.5 and 6.25% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 1.6 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
The stock solutions and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test System and Supporting Information
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.
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 (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 104 to 105 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

pH:
7.7 - 8.2
Details on test conditions:
Exposure Conditions
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control group and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.55 x 106 cells per mL. Inoculation of 500 mL of test medium with 1.6 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Test Organism Observations
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominal inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
The appearance of the test media was recorded daily.

Comparison of Growth Rates
The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the following equation:
µ = ( lnNn - lnN1 ) / ( tn - t1 )
Where:
μ = average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominal inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
In addition the section by section specific growth rate (Days 0 to 1, 1 to 2 and 2 to 3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:
Ir = ( ( µc - µt ) / µc ) x 100
Where:
Ir = percentage inhibition of average specific growth rate
μc = mean average specific growth rate for the control cultures
μt = average specific growth rate for the test culture

Comparison of Yield
Yield is calculated as the increase in biomass over the exposure period using the following equation:
Y = Nn - N0
Where:
Y = yield
N0 = cell concentration at the start of the test
Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:
Iy = ( ( Yc - Yt ) / Yc ) x 100
Where:
Iy = percentage inhibition of yield
Yc = mean value for yield in the control group
Yt = mean value for yield for the treatment group

Determination of ECx Values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.
Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Statistical Analysis
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
• The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
• The mean of the coefficients of variation of the section by section daily growth rates in the control cultures during the course of the test (Days 0 to 1, 1 to 2 and 2 to 3) must not exceed 35%.
• The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.31 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.63 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Inhibition of Growth Rate
ErC10 (0 to 72 hour): 0.71 mg/L
ErC20 (0 to 72 hour): 1.2 mg/L
ErC50 (0 to 72 hour): 2.8 mg/L*
* It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.
Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences between the control and 0.31 mg/L test concentration (P≥0.05); however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.31 mg/L. Correspondingly, the LOEC based on growth rate was 0.63 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 0.49 mg/L
EyC20 (0 to 72 hour): 0.64 mg/L
EyC50 (0 to 72 hour): 0.99 mg/L; 95% confidence limits 0.89 to 1.1 mg/L
Where EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences between the control and 0.31 mg/L test concentration (P≥0.05); however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 0.31 mg/L. Correspondingly, the LOEC based on yield was 0.63 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 141 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.
Nominal cell density of control at 0 hours : 5.00 x 103 cells per mL
Mean cell density of control at 72 hours : 7.07 x 105 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% 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 cultures over the test period (0 to 72 hours) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 0.31 and 0.63 mg/L. Some enlarged cells were observed to be present in the 1.3 mg/L test cultures and some cell debris was observed in the 2.6 and 5.1 mg/L the test cultures.

Water Quality Criteria
Temperature was maintained at 24 ±1 ºC throughout the test.
The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.2 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 Guideline.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control cultures were observed to be green dispersions. The 0.31 and 0.63 mg/L test cultures were observed to be pale green dispersions, the 1.3 mg/L test cultures were observed to be extremely pale green dispersions whilst the 2.6 and 5.1 mg/L test cultures remained as clear colorless solutions.
Results with reference substance (positive control):
A positive control (Envigo study number YJ31TQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted between 12 November 2018 and 03 December 2018.
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 to 72 hour) : 1.5 mg/L; 95% confidence limits 1.3 to 1.7 mg/L
EyC50 (0 to 72 hour) : 0.76 mg/L; 95% confidence limits 0.69 to 0.85 mg/L
No Observed Effect Concentration based on growth rate: 0.50 mg/L
No Observed Effect Concentration based on yield: 0.50 mg/L
Lowest Observed Effect Concentration based on growth rate: 1.0 mg/L
Lowest Observed Effect Concentration based on yield: 1.0 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item. In-house data from the last 5 years shows a mean ErC50 value of 1.4 mg/L (standard deviation = 0.26) and a mean EyC50 value of 0.66 mg/L (standard deviation = 0.14).

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 over a 72 hour period and based on the 0-Hour measured test concentrations gave the following results:
Growth rate
EC50 = 2.8 mg/L
NOEC = 0.31 mg/L
LOEC = 0.63 mg/L

Yield
EC50 = 0.99 mg/L (95%CL: 0.89-1.1)
NOEC = 0.31 mg/L
LOEC = 0.63 mg/L