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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 -10 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Specific details on test material used for the study:
99.3 % MOTE
0.7 % DOTE
0.2 % EHTG
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 item, the test concentration analysis were based on the nominal concentrations of the test item (in the form of test item 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 substance 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
Details on test solutions:
Based on the in house dissolution test, DOTE 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 item formulation.
OECD alga culture medium was used for pre-culture and as the diluent medium (refers Appendix 11 for the procedure for preparation of OECD alga medium and Appendix 12 for dates of preparation and expiry).
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:
0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L
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 dose ranging study and limit test respectively to use in the test culture as inoculum. 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 (Appendix 7 and 8). 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 23.1 (on day 3) during range finding study and 11.2 (on day 2) and 22.6 (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 chosen concentration was prepared by dilution of stock solution. Stock solution was prepared by weighing required quantity of test item in beaker, to this required volume of DMSO (100 µL/L) was added and stirred well using glass rod, after the complete miscible of test item, required volume of OECD alga media was added and stirred well using glass road after complete miscible, volume was transferred to measuring cylinder, beaker was rinsed with OECD alga media and transferred again to measuring cylinder, rinsing procedure was repeated until the complete transfer of contents. Finally, volume was made up to required volume using OECD alga media. The prepared test formulation was kept on magnetic stirrer to maintain the homogeneity. Prepared solution was used as stock solution. From this stock solution, required volume of stock solution was taken and diluted to required volume to make desired test concentrations both during range finding study and limit test (Appendix 9 and 10).
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 5610 and 5676 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:
The positive control has not been included in this study as the reliability of the Alga Growth Inhibition was tested using 3, 5 Dichloro phenol as a part of the Study No.: BIO-ET 052. The positive control result was incorporated in the study report
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:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate

Algal Cell Count and Observations

The alga cells were found healthy in both control and treatment groupsand there were no treatment-related changes observed in the algal cell counts at 24, 48 and 72 hours during the 72 hour exposure period.

Refer: Table 1 to 4; Appendix 1 to 2

Environmental Parameters

Light intensity and temperature were recorded daily. Test flask containing only alga culture medium (blank) served as a surrogate for the temperature measurement during pre-culture (acclimation) and exposure regimes.

Light intensity of 5134 to 5352 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 was ranged from 8.10 to 8.23 at the beginning (0 h) and 8.01 to 8.21 at the end of the test (72 h) in the range finding study. During limit test pH of the test concentrations was ranged from 8.10 to 8.30 at the beginning (0 h) and 8.07 to 8.27 at the end of the test (72 h).

Refer:Appendix 3 to 6

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 MOTE, respectively. This was comparable with a control (solvent) growth rate of 1.37%.

Refer: Table 5

During the limit test an average specific growth rate of 1.38% was observed in the tested concentration of 100.0 mg/L of DOTE. This was comparable with the control (solvent) growth rate of 1.40%.

Refer: Table 6

Percent Inhibition in Growth Rate

At 72 hours percent growth rate inhibition of 0.97, 0.24, 0.24, 0.00, 0.00 and 0.48 % was observed in the treated concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L of MOTE, respectively, during the range finding study when compared to the control (solvent).

Refer: Table 5

 

In the limit test, at 72 hours a percent growth rate inhibition of 1.43% was observed in the tested concentration of 100.0 mg/L of MOTE, as compared to the controls.

 

Refer: Table 6

Percent 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 concentration of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L of MOTE, respectively during range finding study as compared with the control (solvent).

Refer: Table 7

In the limit test a percent inhibition in algal yield of 5.06 % was observed at the test the test concentration of 100.0mg/L of MOTE.

Refer: Table 8

Measurement and analytical determinants

During the range finding sludy and limit test, sample from all the test item concentration collected at 0 hour was analyzed for the concentration and sample collected at 72 hour was analyzed for the stability, results reported that test concentrations at the 0 hour and stability at 48 hour were in the acceptable range of ± 20% recovery to the nominal concentrations.

Refer: Table 11 & 12, Appendix 13

Results of Reference Standard

The reference standard study (BIO-ET 052), 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.

Statistical Analysis

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 experimental conditions employed, it can be concluded that the growth rate ErC50 and inhibition in the yield EyC50 for the test item MOTE was greater than 100.0 mg/L.
The No Observed Adverse Effect Concentration (NOAEC) over the 72 hours exposure period was also greater than 100.0 mg/L of MOTE.
Executive summary:

The test item was tested for Alga (Pseudokirchneriella subcapitata) Growth Inhibition in accordance with OECD Guidelines for Testing of Chemicals (Section 2), Effects on Biotic Systems, Guideline No. 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” adopted on 23 March 2006. 

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.

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 alga cells (5610 cells/mL) were exposed to a range of selected concentration. At 72 hours the Percent Growth Rate Inhibitions of 0.97, 0.24, 0.24, 0.00, 0.00 and 0.48 % were observed at the treated concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L of MOTE, respectively, as compared with the solvent control.

Based on the results of the range finding study, a limit test was conducted as the main study at a MOTE 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 (5676 cells/mL) were exposed to range of selected concentrations. 

A Percent Growth Rate Inhibition of 1.43 % was observed at the treated concentration of 100.0 mg/L of MOTE, as compared to the control (solvent). Similarly, a Percent Inhibition in Algal Yield of 5.06 % was observed at the MOTE concentration of 100.0 mg/L as compared with the control (solvent).

This algal growth inhibition study fulfills all the validity criteria of the test. The control culture increased exponentially by a factor of 60.14 and 66.38 during 72 hour test period, mean coefficient of variation for section-by-section specific growth rates were 33.04 and 31.54 %, and the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 0.73 and 0.29 in the range finding and limit tests, respectively. These results support the conclusion that the test is valid.

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

Reference standard study (BIO-ET 052), with 3, 5 Dichloro phenol obtained growth rate inhibition ErC50 of 3.41 mg/L and inhibition in yield EyC50was 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 test procedures followed.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
March 16 to March 19, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The test material only contained 70% MOTE and in addition the substance tested is believed to contain EHTG as an impurity, which is more soluble than the registered substance and known to be more hazardous. This technical grade of the material is not considered to be the most representative of MOTE itself and therefore the effects noted are not considered to be representative of the registered substance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Duplicate samples for analysis were taken from the highest test concentration and the control according to the schedule below. The method of analysis is described in the appended Analytical Report.

Frequency at t=0 h, t=24 h and t=72 h
Volume 25 ml
Storage Not applicable, samples were analysed on the day of sampling.
Vehicle:
no
Details on test solutions:
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g. film of the test substance on the water surface).

The batch of Octyltintris(2-ethylhexyl mercaptoacetate) 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/I applying one day of magnetic stirring followed by a one day stabilisation period to reach maximum solubility of the test substance 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.

Alter preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10*4 cells/ml.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Pseudokirchneriella subcapitata, strain: NIVA CHL 1

Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/l
Test temperature:
22.7 to 23.1 °C
pH:
6.0-9.0, preferably not varying by more than 1.5 units
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
1.0, 10 and 100% of a WSF prepared at a loading rate of 100 mg/I
Details on test conditions:
Test duration: 72 hours
Test type: Static
Test vessels: 100 ml, all-glass, containing 50 ml of test solution
Medium: M2
Cell density: An initial cell density of 1 x 10*4 cells/ml.
Illumination: Continuously using TLD-lamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 85 to 94 µE.m-2.s 1
Incubation: Vessels were distributed at random in the incubator. During incubation the algal cells were kept in suspension by continuous shaking.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: %WSF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 other: %WSF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: %WSF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 100 other: %WSF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Validity criteria fulfilled:
yes
Conclusions:
Due to the very low solubility of Octyltintris(2-ethylhexyl mercaptoacetate) 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/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.
Executive summary:

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 batch of Octyltintris(2-ethylhexyl mercaptoacetate) 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 thetest substance 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 104cells/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 substance 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 (Octyltintris(2-ethylhexyl mercaptoacetate)) 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 Octyltintris(2-ethylhexyl mercaptoacetate) in test medium, concentration levels that might induce >50% reduction of algal growth could not be reached.

The EC50for 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/L. 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.

Description of key information

Sadananda (2016) - Key Study

Under the experimental conditions employed, it can be concluded that  the growth rate ErC50 and inhibition in the yield EyC50 for the test item MOTE was greater than 100.0 mg/L.

The No Observed Adverse Effect Concentration (NOAEC) over the 72 hours exposure period was also greater than 100.0 mg/L of MOTE.

Supporting Studies

Bouwman (2010)

Due to the very low solubility of Octyltintris(2-ethylhexyl mercaptoacetate) 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/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.

Oldersma (2004)

Effect concentrations were based on measured concentrations of the test substance, determined by total Sn (tin) analysis. The NOEC and EC50 (growth rate) were 7 and >445 ug/L MOT(EHMA), respectively. However, the NOEC was based on an exposure concentration of 1.0 mg Sn/L which was extrapolated from higher test concentrations, because the analytical result for the test solution was <LOD.

The best estimate of the mean toxicity of mono-octyltin tris (2-ethylhexylmercaptoacetate) (MOT(EHMA)) to the fresh water alga Scenedesmus subspicatus is the ErC50 of >445 ug MOT(EHMA)/l based on the geometric mean of the measured concentration of MOT(EHMA).

Overall, the studies are in agreement that there are no effects on alagal growth rate at the highest concentration tested (100 mg/L - nominal).

Key value for chemical safety assessment

Additional information

Three relevant studies are available, as follows:

Sadananda (2016) - Key Study

The test item was tested for Alga (Pseudokirchneriella subcapitata) Growth Inhibition in accordance with OECD Guidelines for Testing of Chemicals (Section 2), Effects on Biotic Systems, Guideline No. 201, “Freshwater Alga and Cyanobacteria, Growth Inhibition Test” adopted on 23 March 2006. 

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.

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 alga cells (5610 cells/mL) were exposed to a range of selected concentration. At 72 hours the Percent Growth Rate Inhibitions of 0.97, 0.24, 0.24, 0.00, 0.00 and 0.48 % were observed at the treated concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L of MOTE, respectively, as compared with the solvent control.

Based on the results of the range finding study, a limit test was conducted as the main study at a MOTE 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 (5676 cells/mL) were exposed to range of selected concentrations. 

A Percent Growth Rate Inhibition of 1.43 % was observed at the treated concentration of 100.0 mg/L of MOTE, as compared to the control (solvent). Similarly, a Percent Inhibition in Algal Yield of 5.06 % was observed at the MOTE concentration of 100.0 mg/L as compared with the control (solvent).

This algal growth inhibition study fulfills all the validity criteria of the test. The control culture increased exponentially by a factor of 60.14 and 66.38 during 72 hour test period, mean coefficient of variation for section-by-section specific growth rates were 33.04 and 31.54 %, and the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 0.73 and 0.29 in the range finding and limit tests, respectively. These results support the conclusion that the test is valid.

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

Reference standard study (BIO-ET 052), with 3, 5 Dichloro phenol obtained growth rate inhibition ErC50 of 3.41 mg/L and inhibition in yield EyC50was 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 test procedures followed.

Supporting Studies

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 batch of Octyltintris(2-ethylhexyl mercaptoacetate) 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 thetest substance 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 10^4 cells/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 substance 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 (Octyltintris(2-ethylhexyl mercaptoacetate)) 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 Octyltintris(2-ethylhexyl mercaptoacetate) in test medium, concentration levels that might induce >50% reduction of algal growth could not be reached.

The EC50for 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/L. 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.

Oldersma (2004)

The summary of mono-octyltin tris (2 -ethylhexylmercaptoacetate) (MOT(EHMA)) to the freshwater green alga Scenedesmus subspicatus was determined in a 72 hour growth inhibition test accroding to OECD 201 and EU C.3 Guidelines. A saturated solution of the test substance (Water Soluble Fraction (WSF)) was prepared with algal growth medium. A test substance concentration series was prepared by repeat dilutions of the WSF and resulted in a testing concentration series of 0, 1.0, 3.2, 9.9, 32 and 99% of the saturated MOT(EHMA) in algal growth medium.

The toxic effect values given below were calculated using the genometric mean of the measured Sn concentrations of the WSF of mono-octyltin tris (2 -ethylhexylmercaptoacetate) (MOT(EHMA)) and the algal cell density in a statistical parametric model. The measured and calculated Sn concentrations were transformed to concentrations of MOT(EHMA) from the tin content of this substance.

The statistical assessment support the conclusion that there was a significant effect only on the growth rate of S. subspicatus in a 72 hour growth inhibition test. It was concluded that the ErC50 value of >63 µg Sn/L (extrapolated 240 µg/L) and >445 µg MOT(EHMA)/L, based on geometric mean of the measured Sn concentrations, represents the best estimate of the median effect concentration of MOT(EHMA) on algal growth. The NOEC in S. subspicatus for MOT(EHMA) is 1µg Sn/L which is equivalent to 7 µg MOT(EHMA)/L. These values are below the LOD of the analytical method of 4.5 µg Sn/L.