Diammonium dihydroxybis[lactato(2-)-O1,O2]titanate(2-)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.07.2018 - 25.01.2019

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)

GLP compliance:

yes

Specific details on test material used for the study:

Batch manufactured & supplied by : Dorf Ketal Chemicals India private Limited
Plot no. 2, Block-F, Sector -12N,
Adani ports & SEZ, Taluka – Mundra,
District-Kutch, Gujarat-370421

Test Item : Tyzor LA

CAS No. : 65104-06-5

Chemical name (IUPAC) : diammonium dihydroxybis[lactato(2-)-O1.O2]titanate(2-)

Titanium in product as TiO2 : 13.71 %

Physical appearance : Clear to slightly hazy.colourless to light yellow liquid

Lot No. : 334-300149/000160

Manufactured date : April 2018

Expiry date : March 2020

Recommended storage condition : Ambient (+15 to +25C)

Specific gravity : 1.215

Analytical monitoring:

not specified

Details on sampling:

The test concentration along with the negative control was analyzed for the test item concentration at the beginning and at the end of test. For analysis, single composite sample was drawn from prepared test concentrations and from the negative control.

Analysis was conducted using a validated analytical method in Analytical R & D Department of Eurofins Advinus Limited. The criteria for acceptance of analysis results of test concentration were 80 to 120 % of claimed concentration with ≤ 20 % RSD of analyzed concentration.

Vehicle:

not specified

Details on test solutions:

Test item stock of 0.1 mg/mL (100 mg/L) was prepared by mixing 50 mg of test item in test medium and making up the final volume to 500 mL in a volumetric flask. To prepare test concentrations of 0.8, 2.4 and 7.2 mg/L, 8, 24 and 72 mL test item stock was mixed separately and volume was made up to 1 L in a volumetric flask using test medium, respectively. Algal cell suspension of 4.3 mL was added before final volume make up for each test concentration.

For negative control (G1), 4.3 mL algal cell suspension was made up to 1 L using test medium.

A stock solution of reference substance (Potassium dichromate- K2Cr2O7) at 1 mg/mL was prepared by mixing 10 mg in 10 mL of test medium. For preparation of reference substance at 0.9 mg/L (G7), 0.9 mL of reference substance stock was made up to 1L using test medium. Algal cell suspension of 4.3 mL was added just before final volume make up.

150 mL of prepared negative control was dispensed into 6 test containers and 150 mL of each test item and reference substance concentration was dispensed separately into 3 test containers.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata ATCC 22662

This algal species was used as the test system as it is a recommended algal species as per the guideline.
6.1.2.1 Source
American Type Culture Collection
P.O.Box 1549
Manassas, VA 20108
USA
6.1.2.2 Culture Maintenance
Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between 21 and 25°C (±2°C) under a continuous illumination of 8000±1600 Lux.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

not specified

Test temperature:

21.8 to 22.3 C

pH:

7.77 to 7.82

Dissolved oxygen:

not specified

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

0.8 , 2.4 , 7.2 , 22 , 66 mg/L

Details on test conditions:

6.2.1 Outline of the Method
Exponentially growing cultures of unicellular green alga Pseudokirchneriella subcapitata were exposed to various concentrations of Tyzor LA, over several generations under defined conditions. The inhibition of growth in relation to a negative control culture was determined over a period of 72 hours.
6.2.2 Preparation of Inoculum Culture
To adapt and to ensure that the algae are in the exponential growth phase, inoculum culture was prepared by inoculating Pseudokirchneriella subcapitata of continuous/mother culture to test medium 4 days before the start of the test. The flasks were incubated inside the algal growth chamber under the test conditions. The cell density of inoculum culture was 104.5x104 /mL during range finding test and 238x104 /mL during definitive test and appropriate volume were used to achieve cell density of around 1x104cells/mL in final test solution.
6.2.3 Test Conditions
Algal cells with test concentrations were incubated in the following conditions during range finding and the definitive test. The test containers were plugged using cotton and randomly placed in the growth chamber. Test containers were repositioned every day during the treatment period

Parameters Range finding test Definitive test
Duration of the test 72 ± 1 hours 72 ± 1 hours
Test containers 250 mL volumetric flask 250 mL volumetric flask
pH of test concentration 7.73 to 8.42 7.77 to 7.82
Temperature of growth chamber (C) 22.2 to 22.4 21.8 to 22.3
Mean continuous light intensity (Lux) 6910 to 7050 6475 to 6613
6.2.4 Test Item Solubility
Tyzor LA was tested for its solubility by mixing 1000 mg of test item and making up the volume to 10 mL in a volumetric flask using test medium.
6.2.5 Range Finding Test
Range finding test was conducted to select the test concentrations for the definitive test with a negative control and test item concentrations at 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L. Each group had single replicate.
6.2.6 Definitive Test
Based on the results of the range finding test, the definitive test was carried out using test concentrations of 0.8 (G2), 2.4 (G3), 7.2 (G4), 22 (G5) and 66 (G6) mg/L (factor of 3) along with a negative control and a reference substance.
6.2.6.1 Preparation of Test Concentrations
Test item stock of 0.1 mg/mL (100 mg/L) was prepared by mixing 50 mg of test item in test medium and making up the final volume to 500 mL in a volumetric flask. To prepare test concentrations of 0.8, 2.4 and 7.2 mg/L, 8, 24 and 72 mL test item stock was mixed separately and volume was made up to 1 L in a volumetric flask using test medium, respectively. Algal cell suspension of 4.3 mL was added before final volume make up for each test concentration.

For negative control (G1), 4.3 mL algal cell suspension was made up to 1 L using test medium.

A stock solution of reference substance (Potassium dichromate- K2Cr2O7) at 1 mg/mL was prepared by mixing 10 mg in 10 mL of test medium. For preparation of reference substance at 0.9 mg/L (G7), 0.9 mL of reference substance stock was made up to 1L using test medium. Algal cell suspension of 4.3 mL was added just before final volume make up.

150 mL of prepared negative control was dispensed into 6 test containers and 150 mL of each test item and reference substance concentration was dispensed separately into 3 test containers.
6.2.6.2 Analysis of Test Concentrations
The test concentration along with the negative control was analyzed for the test item concentration at the beginning and at the end of test. For analysis, single composite sample was drawn from prepared test concentrations and from the negative control.

Analysis was conducted using a validated analytical method in Analytical R & D Department of Eurofins Advinus Limited. The criteria for acceptance of analysis results of test concentration were 80 to 120 % of claimed concentration with ≤ 20 % RSD of analyzed concentration.
6.2.7 Observations
Following parameters were observed and recorded during both range finding test and definitive test.

• The pH of the controls and the test solutions was checked at the beginning and at the end of the test.
• Light intensity inside the algal chamber (at four random locations) was measured once daily using a Lux meter.
• Temperature inside the algal chamber was recorded once daily.
• The algal biomass in each flask was determined at 24, 48 and
72 hours after the start of the test using a haemocytometer.
• The measured cell concentrations in the test cultures and controls were tabulated together with the concentrations of the test item and the times of measurement.

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32.707 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

9.1 Test Item Solubility
The test item formed a miscible in test medium at the tested concentration of 100 mg/mL.
9.2 Range Finding Test
Test conditions are presented in Table 1.

Summary of average algal cell biomass is presented in Table 2.

The reduction of algal cell biomass at 72 h exposure at the tested concentration of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L was 0.35, 0, 2.12, 4.24, 4.95, 25.44, 50.88 and 93.64%, respectively.
9.3 Definitive Test
Test conditions are presented in Table 3.

Summary of average algal cell biomass, percent growth rate inhibition, percent yield inhibition, average specific growth rate are presented in Table 4 to 7.

Growth curve of algal cell biomass, inhibition of growth rate and yield, percent inhibition of cell growth rate, per cent inhibition of yield, dose response curve of growth rate and dose response of yield are presented in Figures 1 to 6, respectively.

At test concentration of 0.8, 2.4, 7.2, 22 and 64 mg/L, the observed inhibition in the percent growth rate of algal biomass was 2.6, 8.38, 14.02, 26.87 and 78.53 %; and the percent yield inhibition was 10.60, 30.49, 45.65, 69.21 and 97.87 %, respectively when compared to that of the negative control during the test period.

There was 19.35 % and 57.02 % inhibition of average growth rate and yield of alga, respectively, in the reference substance group at 0.9 mg/L when compared to negative control. Observed cells were found morphologically normal.

The calculated EC50 value at 72 hours for growth rate was 32.707 mg/L, with fiducial limits being 14.4725 and 213.7741 mg/L.

The calculated EC20 value at 72 hours for growth rate was 8.5808 mg/L, with fiducial limits being 3.79692 and 86.08445 mg/L.

The calculated EC10 value at 72 hours for growth rate was 4.2632 mg/L, with fiducial limits being 1.88643 and 27.86449 mg/L.

The calculated EC50 value at 72 hours for yield was 7.125 mg/L, with fiducial limits being 3.3781 and 15.9929 mg/L.

The calculated EC20 value at 72 hours for yield was 1.7727 mg/L, with fiducial limits being 0.84051 and 3.97917 mg/L.

The calculated EC10 value at 72 hours for yield was 0.8567 mg/L, with fiducial limits being 0.40618 and 1.92294 mg/L.
9.4 Analysis of Test Concentrations
Summary of analysis of test concentrations on day of preparation and end of definitive test and recovery of test item in test medium is presented in Table 8 to 10.

The test item was recoverable at concentrations of 0.160 and 106 mg/L in the test medium.

The active ingredient concentration analysis in all test concentrations showed that the per cent agreement with claimed concentration was 99.18 to 110.3% at the start of test and 88.99 to 102.1% at end of test (72 hour) ) indicating that the results were within the acceptable limit (80 to 120 % of the claimed concentration with an RSD of < 20%). No measurable concentration of test item was recorded in the control samples.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 value for growth rate and yield on the basis of the nominal concentration of the test item are presented below:

At 72 hours EC values (mg/L)
ErC50 32.707
ErC20 8.5808
ErC10 4.2632
EyC50 7.125
EyC20 1.7727
EyC10 0.8567

Note: ErC refers to growth rate; EyC refers to yield.

Executive summary:

The effect of Tyzor LA was tested on the growth of fresh water single cell green algaPseudokirchneriella subcapitata.

 

The alga was exposed to the test item at the nominal concentrations of 0.8, 2.4, 7.2, 22 and 64 mg/L (factor of about 3) along with a negative control and reference substance. The cell growth was measured at 24, 48 and 72 hours after the initiation of the test.

 

The concentration/effect relationship was determined using two factors namely growth rate and yield at the end of the test (72 hours). The inhibition of growth rate was 2.6, 8.38, 14.02, 26.87 and 78.53 % and the inhibition of yield was 10.60, 30.49, 45.65, 69.21 and 97.87 % at the tested concentrations of 0.8, 2.4, 7.2, 22 and 64 mg/L, respectively, when compared to the negative control. There was 19.35 % and 57.02 % inhibition of average growth rate and yield of alga, respectively, in the reference substance group at 0.9 mg/L when compared to negative control. Observed cells were found morphologically normal.

 

The test item was recoverable at concentrations of 0.106 and 106 mg/L in the test medium. The active ingredient concentration analysis in all test concentrations showed that the per cent agreement with claimed concentration was 99.18 to 110.3% at the start of test and 88.99 to 102.1% at end of test (72 hour))indicating that the results were within the acceptable limit (80 to 120 % of the claimed concentration with an RSD of<20%).

 

The EC₅₀value for growth rate and yield on the basis of the nominal concentration of the test item are presented below:

 

At 72 hours	EC values (mg/L)
ErC50	32.707
ErC20	8.5808
ErC10	4.2632
EyC50	7.125
EyC20	1.7727
EyC10	0.8567

 

Note: E_rC refers to growth rate; E_yC refers to yield.

Description of key information

The EC₅₀value for growth rate and yield on the basis of the nominal concentration of the test item are presented below:

 

At 72 hours	EC values (mg/L)
ErC50	32.707
ErC20	8.5808
ErC10	4.2632
EyC50	7.125
EyC20	1.7727
EyC10	0.8567

 

Note: E_rC refers to growth rate; E_yC refers to yield.

Key value for chemical safety assessment

EC50 for freshwater algae:

32.707 mg/L

EC10 or NOEC for freshwater algae:

4.263 mg/L