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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
Study period:
16 August 2016 to 10 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
other: read-across target
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Algal biomass in each flask was determined daily during the test period. Measurement was made on small volumes removed from the test solution by pipette and volume was not replaced.
Method development for analysis of active content was not practically possible owing to the complexity of the test material. The test concentration analysis was based on the nominal concentrations of the test material (in the form of test material stock solution) introduced into the test medium.
Test concentration analysis was done at Auriga Research Ltd, Unit-III, No. 136, 6th Cross, 2nd Stage, Yeshwanthpur industrial suburb, Bangalore-560022.
Analysis of the concentration of the test material was made at the start (0 hour) and end (72 hour) of all the test concentration during range finding and limit test.
The test concentration samples were collected in duplicate (2 x 10 mL) for each test concentration including vehicle control (negative and solvent) and transferred at ambient condition for test concentration confirmation analysis at Auriga Research Ltd, Unit-III, No. 136, 6th Cross, 2nd Stage, Yeshwanthpur industrial suburb, Bangalore-560022.
Separate test media were prepared specifically for analysis of the exposure concentration during the test, they were treated identically to those used for testing. Algae were separated from medium using centrifugation at a low g-force, sufficient to settle the algae.
Vehicle:
yes
Details on test solutions:
Based on the in house dissolution test, the test material was miscible in DMSO (0.1%) and OECD alga medium. Hence DMSO (0.1%) and OECD alga media was selected as the vehicle for preparation of test material formulation.
OECD alga culture medium was used for pre-culture and as the diluent medium.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Axenic culture of Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), ATCC 22662, obtained from the American Type Culture Collection, was used as the test system for the study.
Alga stock culture was periodically subcultured at least once in a week and maintained with the illumination and temperature of 4440 to 8880 lux and 21 to 24°C, respectively. From this healthy axenic culture of alga, pre culture was prepared of desired cell density to provide the inoculum for test cultures.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
21.1 - 21.6 °C
Nominal and measured concentrations:
Method development for analysis of active content was not practically possible owing to the complexity of the test material. The test concentration analysis was based on the nominal concentrations of the test material (in the form of test material stock solution) introduced into the test medium.
Details on test conditions:
Pre-culture
In order to obtain exponentially growing algal cells, the pre-culture (in two flasks containing 100 mL of culture medium) was initiated 3 days prior to the start of the dose range finding study and limit tests. The algal cell density of pre-cultures (No.1 and 2) during range finding and limit tests were 40650 and 41475 cells/mL at the start, respectively. Algal cell counts were made daily in both the pre-culture flasks. Pre-culture flask No. 2 and Pre-culture flask No.1were used in the range finding and main study, respectively after meeting the acceptable criteria of biomass increase by factor of 10.5 (on day 2) and 22.8 (on day 3) during range finding study and 9.7 (on day 2) and 22.5 (on day 3) during limit test. This acceptance criterion (Biomass increase factor of at least 16) assures that the pre-culture was still in the exponential growth phase prior to be used as inoculum for test cultures.

Test Medium Preparation
The test medium of the chosen concentration was prepared by dilution of the stock solution. The stock solution was prepared by weighing the required quantity of test material in the beaker, to this the required volume of DMSO (100 µL/L) was added and stirred well using a glass rod. The required volume of OECD alga media was added and stirred well using glass rod. Finally, the volume was made up to required volume using OECD alga media. The prepared test formulation was kept on a magnetic stirrer to maintain the homogeneity.
The test media was prepared in the sterile medium by adding exponentially growing culture (inoculum) of Pseudokirchneriella subcapitata. The cells in the culture flasks were maintained in Test media by using an orbital shaker at 100 oscillations per minute during the test period.
The initial alga cell concentrations of the test culture were 5560.5 and 5610 cells/mL in the range finding and limit test, respectively.
There was no evidence of marked change in the pH of the test medium on day 0 of treatment in the range finding and limit test.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Algal Cell Count and Observations
The alga cells were found to be healthy in both control and treatment groups and there were no treatment-related changes observed in the algal cell counts at 24, 48 and 72 hours during the 72 hour exposure period.


Environmental Parameters
Light intensity and temperature were recorded daily. Test flasks containing only algal culture medium (blank) served as a surrogate for the temperature measurement during pre-culture (acclimation) and exposure regimes.
Light intensity of 5134 to 5345 lux was maintained with a universal white type fluorescent lamp during preculture and during range finding and limit test, respectively.
During the test period all the culture flasks were maintained at the test medium temperature of 21.1 to 21.6°C, in the range finding and limit test, respectively. The cells in the culture flasks were maintained in suspension by agitating the test medium continuously at 100 oscillations per minute using an orbital shaker in the range finding and limit test, respectively.
The pH of the test concentrations ranged from 8.10 to 8.20 at the beginning (0 h) and 8.00 to 8.20 at the end of the test (72 h) in the range finding study. During limit test the pH of the test concentrations ranged from 8.10 to 8.31 at the beginning (0 h) and 8.07 to 8.27 at the end of the test (72 h).


Average Specific Growth Rate
Average specific growth rates during the range finding study of 1.36, 1.37, 1.37, 1.38, 1.38 and 1.37% were observed in the test material concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, respectively. This was comparable with a control (solvent) growth rate of 1.38%.
During the limit test an average specific growth rate of 1.37% was observed in the test material concentration of 100.0 mg/L. This was comparable with the control (solvent) growth rate of 1.38%.


Percent Inhibition in Growth Rate
At 72 hours a percent growth rate inhibition of 1.21, 0.49, 0.49, 0.24, 0.24 and 0.72 % was observed in the test material concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, respectively, during the range finding study when compared to the control (solvent).
In the limit test, at 72 hours a percent growth rate inhibition of 0.72% was observed in the test material concentration of 100.0 mg/L, as compared to the controls.
 

Percent Inhibition in Yield
At 72 hours percent inhibition in algal yield of 4.82, 1.61, 2.01, 0.40, 0.40 and 3.21% was observed in the test material concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, respectively during range finding study as compared with the control (solvent).
In the limit test a percent inhibition in algal yield of 1.83% was observed at the test material concentration of 100.0mg/L.


Measurement and analytical determinants
During the range finding study and the limit test, samples from all test concentrations were collected at 0 and 72 hours. These were analysed to determine test material concentration. All concentrations in the range-finder and limit tests were within the acceptable range of ± 20% recovery of the nominal concentration. These results support the conclusion that the test materiral was stable in the test system during the entire 72 hour test period.
Results with reference substance (positive control):
The reference standard study (BIO-ET 052), with 3,5-dichlorophenol obtained growth rate inhibition ErC50 of 3.41 mg/L and inhibition in yield EyC50 was found to be 3.37 mg/L. This 72 hour growth rate inhibition and percent inhibition in yield lies within the validity criteria acceptance range and establishes the acceptability of test system response and confirms the test procedures were followed.
Reported statistics and error estimates:
The response variable in the control and treatment group was analyzed using a statistical Student’s t-test to compare means.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, it can be concluded that the growth rate ErC50 and inhibition in the yield EyC50 for the test material was greater than 100.0 mg/L. The No Observed Adverse Effect Concentration (NOAEC) over the 72 hours exposure period was 100.0 mg/L.
Executive summary:

The toxicity of the test material to the freshwater alga, Pseudokirchneriella subcapitata, was investigated in a study which was conducted in accordance with the standardised guideline OECD 201, under GLP conditions.

A range finding study was conducted with six concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 & 100.0 mg/L along with control (negative and solvent). Each test concentration used 2 replicates; the negative and solvent control groups used 3 replicates. The algal growth (cell density) was assessed at 24, 48 and 72 hours of post-exposure during the test.

During the range finding study exponentially growing algal cells (5560.5 cells/mL) were exposed to a range of selected concentration. At 72 hours the percent Growth Rate Inhibitions of 1.21, 0.49, 0.49, 0.24, 0.24 and 0.72 % were observed at the test material concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, respectively. No growth inhibition was observed in negative or solvent controls during the 72 hour exposure period.

Based on the results of the range finding study, a limit test was conducted as the main study at a test material concentration of 100 mg/L (6 replicates) along with controls (negative and solvent). The algal growth (cell density) was assessed at 24, 48 and 72 hours of post-exposure. Exponentially growing alga cells (5610 cells/mL) were exposed to range of selected concentrations. 

A Percent Growth Rate Inhibition of 0.72 % was observed at the test material concentration of 100.0 mg/L, as compared to the control (solvent).

Analytical results of test material stock solution concentrations at 0 hours and at 72 hours reported that test material stock solution concentration remained at the ± 20% recovery of the nominal concentrations. These results support the conclusion that the test material was stable during the entire 72 hour test period.

Under the conditions of this study, it can therefore be concluded that  the growth rate ErC50 and inhibition in the yield EyC50 for the test material was greater than 100.0 mg/L. The No Observed Adverse Effect Concentration (NOAEC) over the 72 hours exposure period was 100.0 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across from Dioctyltin bis (2-ethylhexylmercaptoacetate) (DOTE) (CAS No. 15571-58-1), see attached justification.
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Description of key information

Read-across from Dioctyltin bis (2-ethylhexylmercaptoacetate) (DOTE) (CAS No. 15571-58-1)

Sadananda, 2016

Under the conditions of this study, it can be concluded that  the growth rate ErC50 and inhibition in the yield EyC50 for the test material DOTE (CAS No 15571-58-1) was greater than 100.0 mg/L. The No Observed Adverse Effect Concentration (NOAEC) over the 72 hours exposure period was 100.0 mg/L of DOTE.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Read-across from Dioctyltin bis (2-ethylhexylmercaptoacetate) (DOTE) (CAS No. 15571-58-1)

Sadananda, 2016

The toxicity of the test material to the freshwater alga, Pseudokirchneriella subcapitata, was investigated in a study which was conducted in accordance with the standardised guideline OECD 201, under GLP conditions.

A range finding study was conducted with six concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 & 100.0 mg/L along with control (negative and solvent). Each test concentration used 2 replicates; the negative and solvent control groups used 3 replicates. The algal growth (cell density) was assessed at 24, 48 and 72 hours of post-exposure during the test.

During the range finding study exponentially growing algal cells (5560.5 cells/mL) were exposed to a range of selected concentration. At 72 hours the percent Growth Rate Inhibitions of 1.21, 0.49, 0.49, 0.24, 0.24 and 0.72 % were observed at the test material concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L, respectively. No growth inhibition was observed in negative or solvent controls during the 72 hour exposure period.

Based on the results of the range finding study, a limit test was conducted as the main study at a test material concentration of 100 mg/L (6 replicates) along with controls (negative and solvent). The algal growth (cell density) was assessed at 24, 48 and 72 hours of post-exposure. Exponentially growing alga cells (5610 cells/mL) were exposed to range of selected concentrations. 

A Percent Growth Rate Inhibition of 0.72 % was observed at the test material concentration of 100.0 mg/L, as compared to the control (solvent).

Analytical results of test material stock solution concentrations at 0 hours and at 72 hours reported that test material stock solution concentration remained at the ± 20% recovery of the nominal concentrations. These results support the conclusion that the test material was stable during the entire 72 hour test period.

Under the conditions of this study, it can therefore be concluded that  the growth rate ErC50 and inhibition in the yield EyC50 for the test material was greater than 100.0 mg/L. The No Observed Adverse Effect Concentration (NOAEC) over the 72 hours exposure period was 100.0 mg/L.