Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 August 2016 to 01 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:
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 complexity of test material, the test concentration analysis were 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 will be done at Auriga Research Ltd, Unit-III, No. 136, 6th Cross, 2nd Stage, Yeshwanthpur industrial suburb, Bangalore-560022.
Analysis of 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 was collected in duplicates (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 was prepared specifically for analysis of exposure concentration during the test, they are 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
Remarks:
dmso
Details on test solutions:
Test Vehicle
- 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.

Test Medium Preoaration
- The test medium was prepared by dilution of stock solution. The stock solution was prepared by dissolving the required quantity of test material in DMSO and making up to volume with OECD alga media. The prepared test formulation was kept on 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.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: ATCC 22662
- Method of cultivation: Alga stock culture was periodically sub-cultured at least once 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.

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 dose range-finding study and limit test respectively. The algal cell density of pre-cultures (No.1 and 2) during range finding and limit test were 40425 and 41850 cells/mL at the start, respectively. Algal cell counts were made daily in both pre-culture flasks. Pre-culture flask No. 1 and Pre-culture flask No.2 were used in the range finding and main study, respectively - pre-cultures met all relevant validity criteria.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
21.1 to 21.6 °C
pH:
8.00 to 8.31
Nominal and measured concentrations:
- Nominal: 100 mg/L
- Measured: 100.1 mg/L (0 hours) and 98.1 mg/L (72 hours)
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterilised, glass conical flasks (Erlenmeyer flasks) of 250 mL capacity
- Fill volume: 100 mL
- The cells in the culture flasks were maintained in suspension by agitating the culture medium continuously at 100 revolutions per minute (rpm) using an orbital shaker.
- Initial cells density: 5610 and 5676 cells/mL in the range finding and limit test, respectively
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Stock solution: 1 (macro nutrients): NH4Cl 1.5 g/L, MgCl2.6(H2O) 1.2 g/L, CaCl2.2(H2O) 1.8 g/L, MgSO4.7(H2O) 1.5 g/L and KH2PO4 0.16 g/L.
- Stock solution: 2 (iron; membrane filtration only): FeCl3.6(H2O) 64 mg/L and Na2EDTA.2(H2O) 100 mg/L.
- Stock solution: 3 (trace elements): H3BO3 185 mg/L, MnCl2.4(H2O) 415 mg/L, ZnCl2 3 mg/L, CoCl2.6(H2O) 1.5 mg/L, CuCl2.2(H2O) 0.01 mg/L and Na2MoO4.2(H2O) 7 mg/L.
- Stock solution: 4 (bicarbonate; membrane filtration only): NaHCO3 50 g/L.
- Stock solutions were sterilised by membrane filtration (mean pore diameter 0.2 µm) or by autoclaving (stock solutions 1 and 3; at 120 °C, 15 min). Stock solutions 2 and 4 were sterilised only by membrane filtration. Stock solutions were stored maximum up to six months in the dark at 4 °C.
- OECD alga growth medium was prepared by adding appropriate volume of the stock solutions 1 - 4 (10 mL of stock solution 1, 1 mL of stock solution 2, 1 mL of stock solution 3, 1 mL of stock solution 4) to 500 mL of sterilised deionised water and made up to 1000 mL. Sufficient time was given for equilibrating the medium with the atmospheric CO2 and pH was set to 8.1.
- Intervals of water quality measurement: The temperature (culture medium) and light intensity (parallel to the level of culture medium) was monitored once in a day during pre culture and exposure. The pH of the test concentrations was measured at the beginning (0 h) and at the end (72 h) of the exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: continuously 5147 to 5352 lux

TEST CONCENTRATIONS
- Based on the results of range finding study the limit test was conducted as main study at the concentration of 100 mg/L along with the control group.
- Range finding study: 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L.

EFFECT PARAMETERS MEASURED:
- The growth of alga was assessed by measuring the cell concentration in each flask at 24, 48 and 72 hours after the start of exposure. Along with the cell counts the alga cell were also observed for their health. The cell density measurements were made on small aliquots sampled aseptically from the test medium by pipette and these samples were discarded after counting.

CELL DENSITY ASSESSMENT
- At each 24 hour interval, cell counts were conducted on each pre-culture and replicate vessel of the treatment and the control groups using a haemocytometer (Neubauer Improved) and microscopes. One sample was removed from each flask for counting. Four haemocytometer fields, each 0.1 × 0.1 cm in size and 0.01 cm deep, containing 0.0001 mL of culture, were examined for algal cells health and counts.
The cell density (cells/mL) in each test flask was calculated for each observation interval by using the formula: Cell Density (cells/mL) = Average cell count × 10000 (CCF) × Dilution factor

AVERAGE SPECIFIC GROWTH RATE
- The average specific growth rate for a specific period was calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:
µi-j = [(ln Xj – ln Xi) /(tj – ti)] (day^-1)
Where,
μi-j is the average specific growth rate from time i to j
Xi is the biomass at time i
Xj is the biomass at time j
For each treatment and control group, mean values for growth rate along with variance estimate was calculated. Average specific growth rate over the entire test duration (days 0-3), using the nominally inoculated biomass as the starting value was calculated. Section-by-section growth rate, calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) was assessed and examined whether the control growth rate remained constant.

PERCENT INHIBITION IN GROWTH RATE
- The percent inhibition of growth rate was calculated for each treatment replicate using the following equation:
%Ir = [(µC - µT) / µC] x 100
Where,
%Ir percent inhibition in average specific growth rate
μC mean value for average specific growth rate (μ) in the control group
μT average specific growth rate for the treatment replicate

PERCENT INHIBITION IN YIELD
- Yield was calculated as the biomass at the end of the test minus the starting biomass for each single vessel of control and treatments. For each test concentration and control, mean value for yield along with variance estimates was calculated. The percent inhibition in yield (%Iy) was calculated for each treatment replicate as follows:
%Iy = [(YC - YT) / YC] x 100
Where,
% Iy percent inhibition of yield
YC value for yield in the control group
YT value for yield for the treatment replicate
Reference substance (positive control):
yes
Remarks:
3, 5 Dichloro phenol
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
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
other: NOAEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield
Details on results:
CELL COUNTS 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.

AVERAGE SPECIFIC GROWTH RATE
- Average specific growth rates during the range-finding study of 1.36, 1.37, 1.37, 1.37, 1.37 and 1.37 % were observed in the tested 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 1.38 % was observed in the tested concentration of 100.0 mg/L. This was comparable with the control (solvent) growth rate of 1.40 %.

PERCENTAGE INHIBITION IN GROWTH RATE
- At 72 hours percent growth rate inhibition of 1.21, 0.49, 0.49, 0.25, 0.25 and 0.73 % was observed in the treated 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 percent growth rate inhibition of 1.19 % was observed in the tested concentration of 100.0 mg/L, as compared with the control.

PERCENTAGE INHIBITION IN YIELD
- At 72 hours the percent inhibition in algal yield of 4.82, 1.61, 2.01, 0.40, 0.40 and 0.77 % was observed in the tested 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 5.06 % was observed in the test the test concentration of 100.0 mg/L.



VALIDITY CRITERIA OF THE TEST
- The biomass in the control cultures increased exponentially by a factor of 60.14 and 66.38 within the 72 hour test period. This corresponds to an average specific growth rate of 1.37 and 1.40 day-1 in the range finding and main study, respectively (validity criterion: biomass should increase at least by factor of 16 and specific growth rate of 0.92 day-1).
- The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3, for 72-hours test) in the control cultures was 32.44 and 31.54 % in the range finding and main study, respectively (validity criterion: must not exceed 35 %).
- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 0.72 and 2.94 in the range finding and main study respectively (validity criterion: should not exceed 7 %).
- Thus the Alga Growth Inhibition Test fulfills the validity criteria of the OECD Guideline No. 201 adopted on 23 March 2006
Results with reference substance (positive control):
The reference standard study, with 3, 5 Dichloro phenol 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.

Table 1: Test Concentration Analysis During the Range-finding Test

 

 Sample

0 Hours

72 Hours

Nominal Content

(g)

% Recovery

Nominal Content

(g)

% Recovery

G1

0.0

-

0.0

-

G2*

0.0

-

0.0

-

G3

0.01

100

0.01

94.8

G4

0.1

100

0.1

97.1

G5

1.0

92

1.0

89.4

G6

10.0

90

10.0

88.8

G7

50.0

100.58

50.0

96.4

G8

100.0

101.33

100.0

97.2

Table 2: Test Concentration Analysis During the Limit Test

 

 

Sample

0 Hours

72 Hours

Nominal Content

(g)

% Recovery

Nominal Content

(g)

% Recovery

G1

0.0

-

0.0

-

G2*

0.0

-

0.0

-

G3

100.0

100.12

100.0

98.1

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study with Pseudokirchneriella subcapita 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 potential of the test material to cause toxicity to algae was determined in accordance with the standardised guideline OECD 201, under GLP conditions.

The alga cells for both range-finder and definitive studies were found healthy in control and all 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. 

Under the conditions of this study with Pseudokirchneriella subcapita 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.

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 to Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (EC Number 248-227-6 and CAS No 27107-89-7) based on structural similarity and hydrolytical behaviour, 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
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
other: NOAEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
and yield

Description of key information

Read-across to structurally similar substance Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (CAS No 27107-89-7)

Sadananda (2016)

Under the conditions of this study with Pseudokirchneriella subcapita 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.

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 to structurally similar substance Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) (CAS No 27107-89-7)

Sadananda (2016)

The potential of the test material to cause toxicity to algae was determined in accordance with the standardised guideline OECD 201, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The alga cells for both range-finder and definitive studies were found healthy in control and all 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. 

Under the conditions of this study with Pseudokirchneriella subcapita 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.

Bouwman (2010)

The study procedures described in this report were based on the OECD guideline No. 201, 2006. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009, the ISOInternational Standard 8692, 2004 and the OECD series on testing and assessment number 23, 2000. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The batch of test material tested was a clear colourless to light yellow liquid with a purity of 98.00 % and the substance was not completely soluble in test medium at the initial loading rate prepared. Preparation of test solutions started with a loading rate of 100 mg/L applying one day of magnetic stirring followed by a one day stabilisation period to reach maximum solubility of the test material in the test medium. The clear and colourless Water Soluble Fraction (WSF) was filtered through glass wool and used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium.

A combined limit/range-finding test was performed, exposing six replicates of exponentially growing algal cultures per concentration to the WSF prepared at a loading rate of 100 mg/L and to a control group. In addition, three replicates of exponentially growing algal cultures per concentration were exposed to 1.0 and 10 % of the WSF. The initial cell density was 10^4cells/mL and the total test period was 72 hours. Duplicate samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test. Analysis was performed on the octyltin species in Octyltintris(2-ethylhexyl mercaptoacetate). The measured concentration of octyltin species in the duplicate samples taken from the undiluted WSF showed initial concentrations of 18 and 27 µg/L. These concentrations decreased to 37 % of initial after 24 hours of exposure and further to 23 % of initial at the end of the test. The decrease of test material concentrations might be explained by initial concentrations being above the solubility limit. This explanation was supported by the variance between duplicate samples taken at the start of the test. Given these results, the effect parameters were expressed in terms of both loading rate of the test material and the Time Weight Average (TWA) concentration (octyltin species). The TWA concentration was calculated to be 8.8 µg/L.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Due to the very low solubility of the test material in test medium, concentration levels that might induce >50% reduction of algal growth could not be reached. The EC50 for both growth rate reduction (ERC50: 0-72h) and yield inhibition (EyC50: 0-72h) was beyond the concentration obtained in a WSF prepared at a loading rate of 100 mg/I. The concentration of organotin species measured in this WSF was 8.8 µg/L. The NOEC for growth rate reduction equalled the concentration obtained in a WSF prepared at a loading rate of 100 mg/L, while the NOEC for yield inhibition was below this concentration.