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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 August 2016 - 10 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 24oC, 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 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.
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 40650 and 41475 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 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 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 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:
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

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 5345 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 21.6oC, 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 8.20 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 pH of the test concentrations was 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).

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, 1.38, 1.38 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 DOTE, respectively. This was comparable with a control (solvent) growth rate of 1.38%..

Refer: Table 5

During the limit test an average specific growth rate of 1.37% was observed in the tested concentration of 100.0 mg/L of DOTE. This was comparable with the control (solvent) growth rate of 1.38%.

Refer: Table6

Percent Inhibition in Growth Rate

At 72 hours percent growth rate inhibition of 1.21, 0.49, 0.49, 0.24, 0.24 and 0.72 % was observed in the treated concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L of DOTE, 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 0.72% was observed in the tested concentration of 100.0 mg/L of DOTE, 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 3.21 % was observed in the tested concentration of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L of DOTE, 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 1.83 % was observed at the test the test concentration of 100.0mg/L of DOTE.

Refer: Table 8

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 analyzed to determine DOTE concentration. All concentration 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 nominal test concentrations of DOTE at 0 hour were confirmed and the concentrations of DOTE at 72 hour reflected stability of the DOTE test system during the entire 72 hour test period. s.

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 ErC50of 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 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:
Based on the above results of the experiment with Pseudokirchneriella subcapita and under the experimental conditions employed, it can be concluded that the growth rate ErC50 and inhibition in the yield EyC50 for the test item DOTE 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 DOTE.
The United Nations Globally Harmonized System [GHS] sets classification criteria for the 72-hour aqueous ErC50 in algae [e.g. P. subcapita]. The ErC50 and EyC50 of DOTE in P. subcapita are greater than 100 mg/L and the current GHS criteria indicate that a classification category would not be assigned to DOTE. A substance with ErC50 and EyC50 >100 mg/L would be designated as Not Classified for hazards to the aquatic environment.
Executive summary:

 The test item dioctyltin bis-ethylhexylthioglycolate [DOTE] was obtainedfrom Galata Chemicals LLC. It 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 (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 treated concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L ofDOTE, respectively.No growth inhibition was observed in negative or solvent controls during the 72 hour exposure period.

Similarly, at 72 hours the percent inhibition in algal yield of 4.82, 1.61, 2.01, 0.40, 0.40 and 3.21 % was observed at the tested concentrations of 0.01, 0.1, 1.0, 10.0, 50.0 and 100.0 mg/L ofDOTE, respectively. No yield 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 DOTE 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 treated concentration of 100.0 mg/L ofDOTE, as compared to the control (solvent). Similarly, a Percent Inhibition in Algal Yield of 1.83 % was observed at the DOTE 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 60.98 and 63.96 during 72 hour test period, mean coefficient of variation for section-by-section specific growth rates were 34.24 and 28.85 %, and 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 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 DOTE at 0 hour were confirmed and that concentrations of DOTE were stable during the entire 72 hour test period.

Reference standard study (BIO-ET 052), with 3, 5 Dichloro phenol obtained growth rate inhibition ErC50of 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.

Description of key information

Based on the above results of the experiment with Pseudokirchneriella subcapita and under the experimental conditions employed, it can be concluded that the growth rate ErC50 and inhibition in the yield EyC50 for the test item DOTE 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 DOTE.

The United Nations Globally Harmonized System [GHS] sets classification criteria for the 72-hour aqueous ErC50 in algae [e.g. P. subcapita]. The ErC50 and EyC50 of DOTE in P. subcapita are greater than 100 mg/L and the current GHS criteria indicate that a classification category would not be assigned to DOTE. A substance with ErC50 and EyC50 >100 mg/L would be designated as Not Classified for hazards to the aquatic environment.

hours exposure period was also greater than 100.0 mg/L of DOTE.

The United Nations Globally Harmonized System [GHS] sets classification criteria for the 72-hour aqueous ErC50in algae [e.g.P. subcapita]. The ErC50andEyC50of DOTE inP. subcapitaaregreater than 100 mg/Land the current GHS criteria indicate that a classification category would not be assigned to DOTE. A substance with ErC50andEyC50 >100 mg/L would be designated as Not Classified for hazards to the aquatic environment.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L

Additional information

There are two additional studies summarised for this endpoint; both were conducted under GLP conditions.

Both studies were conducted with a test item containing ca. 3 -4 % of Ethylhexylthioglycolate (EHTG, CAS: 7659 -86 -1, EC: 231 -626 -4) as impurity

EHTG is better soluable in water as DOTE and shows the following ecotoxicity:

EC50 (48 h) Daphnia = 0.38 mg/L

EC50 Algae = 0.41 mg/L

and is thus classified as Aquatic Acute 1 H400 and Aquatic Chronic H410

Is likely that the observed effects in both studies are caused or exacerbated by the impurity