Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
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 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
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, 1.0, 2.2, 4.6, 10, 22, 46, 100% of Saturated Solution
- Sampling method: 2.0 mL from the approximate centre of the test vessels, at t=0h and t=48h.
- 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 were pooled at each concentration before sampling.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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 in the combined limit/range-finding test and two hours and a half in the full test. 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):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Daphnia magna Straus 1820
- Source: In-house laboratory culture with a known history.
- Age of parental stock: >2 weeks
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Feeding during test: no
- Age of test organisms at start of test: <24 h

BREEDING
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.
- Medium: M7 medium

Study design

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

Test conditions

Hardness:
180 mg/L expressed as CaCO3
Test temperature:
20°C
pH:
start: 7.9
end: 7.9-8.0
Dissolved oxygen:
start: 8.9-9.1
end: 8.9-9.0
Nominal and measured concentrations:
Nominal: 0, 1.0, 2.2, 4.6, 10, 22, 46, 100 % of Saturated Solution
Measured at start: n.d., 0.198, 0.442, 0.985, 2.05, 4.64, 10.2, 22.4 mg/L
Measured at end: n.d., 0.209, 0.472, 0.983, 2.06, 4.58, 10.1, 21.6 mg/L
The measured concentrations remained stable throughout the test duration, i.e. were 97-107% of initially measured at the end of the test. Given these results, the EC50 was calculated using the measured initial concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL all-glass, containing 50 mL test solution
- Aeration: no aeration
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Feeding: no feeding during test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, made with Reverse Osmosis water with the following chemicals dissolved: CaCl2.2H2O (211.5 mg/L), MgSO4.7H2O (88.8 mg/L), NaHCO3 (46.7 mg/L) and KCl (4.2 mg/L).
- Culture medium different from test medium: yes (M7 medium used as culture medium)
- Intervals of water quality measurement: Dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control. In addition, after 24 hours, immediately after counting the immobilized daphnids, oxygen levels were measured in one replicate of the undiluted SS because all the Daphnia magna were immobilized. Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours photoperiod daily.

EFFECT PARAMETERS MEASURED:
- Immobility (including mortality), at 24 h and at 48 h.

RANGE-FINDING STUDY
- Test concentrations: control (0%), 1.0, 10, 100% of Saturated Solution 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
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
13 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
- After 24 hours of exposure, 100% effect was observed in the highest concentration, while no biologically significant immobility was recorded at any of the lower concentrations tested and in the control. After 48 hours of exposure, a dose-response relationship was observed.
- Behavioural abnormalities: None observed.
- Mortality of control: None observed.
- Other adverse effects control: No signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
Results with reference substance (positive control):
- Results with reference substance valid? Yes. The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.
- Relevant effect levels:
24h-EC50: 0.95 mg/L with a 95% confidence interval between 0.85 and 1.1 mg/L.
48h-EC50: 0.59 mg/L with a 95% confidence interval between 0.52 and 0.66 mg/L.
Reported statistics and error estimates:
The 24 and 48h-EC50-values were calculated using the Spearman-Karber method (without trim) on the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (measured initial).
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)

Replicate

Tri butyl thiourea; measured initial concentrations (mg/L)

Control

0.20

0.44

0.98

2.0

4.6

10

22

0

A

5

5

5

5

5

5

5

5

B

5

5

5

5

5

5

5

5

C

5

5

5

5

5

5

5

5

D

5

5

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

20

20

 

 

24

A

0

0

0

0

0

0

0

5#

B

0

0

0

0

0

0

0

5

C

0

0

0

2

0

0

0

5

D

0

0

0

0

1

0

1

5

Total immobilised

0

0

0

2

1

0

1

20

Effect %

0

0

0

10

5

0

5

100

 

 

 

 

 

 

 

 

48

A

0

0

0

1

1

2

5

5

B

0

0

0

0

2

2

5

5

C

0

0

0

3

1

2

5

5

D

0

0

0

1

3

3

5

5

Total immobilised

0

0

0

5

7

9

20

20

Effect %

0

0

0

25

35

45

100

100

#Microscopic observation revealed no test item stuck to the daphnids.

Analysis of Test Samples (Final Test)

Time of sampling
[hours]

Date of sampling

Date of
analysis1

Percentage of SS2
[%]

Analyzed concentration
[mg/L]

Relative to
initial
[%]

0

07 Aug 2017

21 Aug 2017

0

n.d.

 

 

 

 

1.0

0.198

 

 

 

 

2.2

0.442

 

 

 

 

4.6

0.985

 

 

 

 

10

2.05

 

 

 

 

22

4.64

 

 

 

 

46

10.2

 

 

 

 

100

22.4

 

48

09 Aug 2017

21 Aug 2017

0

n.d.

n.a.

 

 

 

1.0

0.209

105

 

 

 

2.2

0.472

107

 

 

 

4.6

0.983

100

 

 

 

10

2.06

101

 

 

 

22

4.58

99

 

 

 

46

10.1

99

 

 

 

100

21.6

97

1     Samples were stored in the freezer (≤ -15°C) until the day of analysis.

2     Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

n.d.      Not detected.

n.a.Not applicable.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For details on validity criteria see 'Overall remarks' section.
Conclusions:
In conclusion, the 48h-EC50 for Daphnia magna exposed to Tri butyl thiourea was 3.0 mg/L based on measured initial concentrations (95% confidence interval between 2.2 and 4.0 mg/L).
Executive summary:

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to Tri butyl thiourea at nominal concentrations of 0, 1.0, 2.2, 4.6, 10, 22, 46, and 100% of a Saturated Solution prepared at 100 mg/L (measured initial concentrations: n.d., 0.20, 0.44, 0.98, 2.0, 4.6, 10, 22 mg/L). A dose-response relationship for immobility was observed at 48 h. The 48h-EC50 for Daphnia magna exposed to Tri butyl thiourea was 3.0 mg/L based on measured initial concentrations. The study is considered to be reliable without restrictions.