Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jul 2017 - 21 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
water solubility
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 22 March 2017 to 01 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
EU Method A.6 (Water Solubility)
Version / remarks:
2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 105 (Water Solubility)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 830.7840 (Water Solubility)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Type of method:
flask method
Key result
Water solubility:
0.016 g/L
Incubation duration:
>= 24 - <= 168 h
Temp.:
20 °C
pH:
>= 6.3 - <= 7
Details on results:
The 72-hour measurement was significantly higher than the 24- and 48- hour measurements (maximum difference > 15%). In order to rule out any erroneous sample preparation of the deviating sample from the 72-h-stirred vessel, this sample was reanalysed in duplicate. Obtained results, however, were comparable to what was obtained during initial analysis.

As observed concentrations increased with stirring time, stirring was prolonged. The 168-hour measurement was, again, significantly higher than the 120- and 144- hour measurements (maximum difference > 15%). Obviously the higher result of the third vessel was linked to the specific water solubility vessel. No observations had been made which could explain the difference in observed concentrations. 120-, 144- and 168-hour measurements illustrated, however, that the equilibrium concentration was already reached. The coefficient of variation obtained for the different water solubility concentrations was 22%. As this approximated what was considered acceptably repeatable during validation, the water solubility of the test item was calculated as the mean value of all measurements.

 Results of the flask method

Stirring time
[hours]

Analyzed concentration
[g/L]

Mean
[g/L]

Maximum difference
[%]

pH

24

0.0136

 

 

7.0

48

0.0149

 

 

7.0

72

0.0210

 

 

6.9

72

0.0196

 

 

-

72

0.0187

 

 

-

120

0.0116

 

 

6.5

144

0.0123

 

 

6.4

168

0.0173

 

 

6.3

 

 

 0.016

 58

 

Conclusions:
The water solubility of the substance at 20°C was determined to be 0.016 g/L.
Executive summary:

The water solubility of the substance was determined using the flask method in a GLP study according to EC A.6, OECD 105 and OPPTS 830.7840. Quantification was performed by LC-MS-MS. As observed concentrations increased with stirring time, stirring was prolonged to 168 h. Maximum difference between all results: 58%. The coefficient of variation obtained for the different water solubility concentrations was 22%. As this approximated what was considered acceptably repeatable during validation, the water solubility of the test item was calculated as the mean value of all measurements (24, 48, 72, 120, 144 and 168 hours). The water solubility at 20°C was determined to be 0.016 g/L. The pH of the aqueous samples ranged from 6.3 to 7.0.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Tributyl-2-thiourea
EC Number:
219-350-2
EC Name:
Tributyl-2-thiourea
Cas Number:
2422-88-0
Molecular formula:
C13H28N2S
IUPAC Name:
1,1,3-tributylthiourea
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Tri butyl thiourea
- Appearance: Clear pale to medium brown liquid
- Storage conditions: At room temperature
Specific details on test material used for the study:
Solubility in water: please refer to section 4.8 of this dossier.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, 0.10, 0.32, 1.0, 3.2, 32, 100% of Saturated Solution prepared at 100 mg/L
- Sampling method: 2.0 mL, at t=0 h, t=24 h, and t=72 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration (3.2% SS) but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The batch of Tri butyl thiourea tested was a clear pale to medium brown liquid with a purity of > 97.1% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item. Preparation of test solutions started with a loading rate of 100 mg/L applying a one-day period of magnetic stirring in a closed vessel to ensure maximum dissolution of the test item in medium. The obtained aqueous mixture was allowed to settle for a period of two hours. Thereafter, the Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: Test medium without test item or other additives.
- All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: 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.
- Culture medium different than test medium: yes, M1 medium.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in test medium (M2) at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

ACCLIMATION
- Acclimation period: no

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol/L (24 mg CaCO3/L)
Test temperature:
23°C
pH:
t=0 h: 8.2-8.3
t=72 h: 7.9-8.1
Nominal and measured concentrations:
Nominal: 0.10, 0.32, 1.0, 3.2, 10, 32 and 100% of SS prepared at 100 mg/L.
TWA: 0.000089, 0.00016, 0.074, 0.49, 1.8, 6.4, 18 mg/L
The three highest concentrations remained stable throughout the test duration, all lower concentrations decreased during the test period. Based on these results, the TWA exposure concentrations were calculated and used to determine the effect parameters.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL all-glass, capped, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 284 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 or 2 replicates of each test concentration without algae; 1 extra replicate of each test concentration for sampling purposes after 24 hours of exposure.
- During incubation the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO (tap-water purified by reverse osmosis)
- Culture medium different from test medium: yes, M1.
- Intervals of water quality measurement: pH at beginning and end of test, temperature continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuously
- Light intensity and quality: TLD-lamps with a light intensity within the range of 85 to 86 μE/m2/s.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: algal density was determined by use of a microscope and a counting chamber.
- At the end of the final test microscopic observations were performed on the control and all test concentrations to observe for any abnormal appearance of the algae

TEST CONCENTRATIONS
- Combined Limit/Range-Finding Test concentrations: Control, 1.0, 10, 100% of SS prepared at 100 mg/L.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate (performed June 2017)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Remarks:
Statistically determined
Effect conc.:
0 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Remarks:
Biologically relevant
Effect conc.:
0.074 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.12-0.18
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.28 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.24-0.32
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.94 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 0.86-1.0
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to all test concentrations when compared to the control.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: After 24 hours of exposure during the combined limit/range-finding test, particles were observed at the highest test concentration that disturbed spectrophotometric measurement. No undissolved particles were observed in the replicates of the highest test concentration during the exposure period of the Final Test.
Results with reference substance (positive control):
- Results with reference substance valid? Yes.
- EC50: 72h-EC50 for growth rate inhibition was 0.99 mg/L (95% CI 0.97-1.0 mg/L). The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
Reported statistics and error estimates:
Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller. Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding TWA concentrations of the test item.The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

%SS, Measured and TWA Concentrations

Tri butyl thiourea
% SS at 100 mg/L

Measured concentration (mg/L)

TWA (mg/L)

t=0h

t=24h

t=72 h

0.10

0.0178

0.00000382

0.00000382

0.000089

0.32

0.0612

0.00000382

0.00000382

0.00016

1.0

0.195

0.0983

0.0183

0.074

3.2

0.679

0.519

0.375

0.49

3.21

0.673

0.581

0.473

0.56

10

2.00

1.92

1.66

1.8

32

6.85

6.40

6.09

6.4

100

21.1

17.6

18.0

18

1without algae;2LOD/2 = 0.0000038 mg/L

Growth Rate And Percentage Inhibition For The Total Test Period

Tri butyl thiourea
TWA conc. (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.875

0.0783

6

0.000089

1.874

0.0662

3

0.03

0.00016

1.739

0.0237

3

7.25#

0.074

1.723

0.0910

3

8.11#

0.49

1.241

0.0662

3

33.83*

1.8

0.717

0.1268

3

61.76*

6.4

0.025

0.0429

3

98.68*

18

0.000

0.0000

3

100.00*

* effect was statistically significant;#effect was statistically significant but biologically not relevant (<10%)

Effect Parameters

Parameter (mg/L)

NOEC
(stat)*

NOEC
(biol)#

EC10

EC20

EC50

Growth rate

Value

0.000089

0.074

0.15

0.28

0.94

lower 95%-cl

 

 

0.12

0.24

0.86

upper 95%-cl

 

 

0.18

0.32

1.0

Yield

Value

0.000089

0.000089

0.000050

0.00036

0.015

lower95%-cl

 

 

0.000018

0.00016

0.0093

upper95%-cl

 

 

0.00012

0.00070

0.024

*Statistically determined NOEC;#Biologically relevant NOEC; cl: confidence limits

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For details on validity criteria see 'overall remarks' section.
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, Tri butyl thiourea inhibited both growth rate and yield of this fresh water algae species at TWA concentrations of 0.00016 mg/L and higher.
The 72h-NOEC for growth rate inhibition was 0.000089 mg/L when based on statistical analysis and 0.074 mg/L when based on biological relevance.
The 72h-EC10 for growth rate inhibition (ERC10) was 0.15 mg/L with a 95% confidence interval ranging from 0.12 to 0.18 mg/L.
The 72h-EC50 for growth rate inhibition (ERC50) was 0.94 mg/L with a 95% confidence interval ranging from 0.86 to 1.0 mg/L.
Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Saturated Solutions of the test substance at Time-Weighted Average concentrations of 0.000089, 0.00016, 0.074, 0.49, 1.8, 6.4, and 18 mg/L. 72h-NOEC for growth rate inhibition was 0.000089 mg/L when based on statistical analysis and 0.074 mg/L when based on biological relevance. The 72h-EC10 for growth rate inhibition was 0.15 mg/L. The 72h-EC50 for growth rate inhibition was 0.94 mg/L. The study is considered to be reliable without resitrictions.