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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2011 - 20 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
During the limit test singular samples for possible analysis were taken from the limit concentration and the control according to the schedule below.
- Frequency: at t=0 h and t=48 h
- Volume: 1.8 ml from the approximate centre of the test vessels
- Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Additionally, reserve samples of 1.8 ml were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface).

The batch of TDI-Urone tested was a clear colourless powder with a purity of 97.5% active content (being a mixture of isomers) and completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with the highest test concentration of 100 mg/l applying a short period of magnetic stirring (~10 minutes) to ensure complete dissolution in test medium. The lower test concentrations for the combined limit/range-finding test were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history.
- Reason for selection: This system has been selected as an internationally accepted invertebrate species.
- 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.
- Age at study initiation (mean and range, SD): For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: no

Method of 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, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
Immobility (including mortality) was measured at 24 hours and at 48 hours.
Post exposure observation period:
Not applicable.
Hardness:
180 mg/l expressed as CaCO3 and the pH: 7.7 ± 0.3.
Test temperature:
The temperature of the test medium was 19.5°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 19.0 and 20.4°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).
pH:
7.6 - 7.8
Dissolved oxygen:
9.2 - 10 mg/L
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal: 100 mg/L (the regulatory limit concentration).
Details on test conditions:
TEST PROCEDURE AND CONDITIONS:
- Test duration: 48 hours
- Test type: Static
- Test vessels: 100 ml, all-glass
- Medium: Adjusted ISO medium
- Number of daphnids: 20 per concentration
- Loading: 5 per vessel containing 80 ml of test solution
- Light 16 hours photoperiod daily
- Feeding: No feeding
- Aeration: No aeration of the test solutions.
- Introduction of daphnids: Within half an hour after preparation of the test solutions.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Not applicable as no effects were observed.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
* The 24h-EC 50 was 0.53 mg/l with a 95% confidence interval between 0.47 and 0.62 mg/l.
* The 48h-EC 50 was 0.28 mg/l with a 95% confidence interval between 0.26 and 0.32 mg/l.
- The actual responses in this reference test with K 2 Cr 2 O 7 were just outside (below) the ranges of the expected historical responses at the different concentrations, i.e. a 48h-EC 50 between 0.3 and 1.0 mg/l. Hence, the sensitivity of this batch of D. magna was slightly higher when compared to the historical data collected at NOTOX.
Reported statistics and error estimates:
Calculation of EC50:
No EC50 could be calculated because the test substance proved to be non-toxic (EC 50 > maximum concentration tested).

RESULTS

1. Calibration curves

Calibration curves were constructed using five concentrations. For each concentration, two responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration^2 weighting factor. The coefficient of correlation (r) was > 0.99 for each curve.

2. Samples

2.1. Procedural recovery samples

The results for the procedural recovery samples are given in Table 1.

Table 1 Procedural recovery samples

Date of preparation [dd-mm-yy] Date of analysis [dd-mm-yy]  Target concentration [mg/l] Nominal concentration [mg/l] Analysed concentration [mg/l] Recovery [%] Mean recovery [%] 
 16-01-12  16-01-12  1 1.00 / 1.00 0.992 / 1.05 99 / 105  102
 16-01-12  16-01-12  100  100 / 100 103 / 102   102 / 101  102
 10-02-12  10-02-12  1  1.00 / 1.00  1.05 / 1.05  105 / 105 105 
 10-02-12  10-02-12  100 100 / 100  102 / 99.5  101 / 99  100

The mean recoveries of the procedural recovery samples fell within the criterion of 70-110%. It demonstrated that the analytical method was adequate for the determination of the test substance in the test samples.

2.2. Test samples

The results for the test samples are given in Table 2 and Table 3.

Table 2 Concentrations of the test substance in test medium - combined limit/range-finding test

Time of sampling [hours] Date of sampling [dd-mm-yy]   Date of analysis 1 [dd-mm-yy]         Concentration Relative to nominal [%]   Relative to initial [%]  
Nominal [mg/l] Analysed [mg/l]
 0  09-01-12  16-01-12  100  101  101  
 48  11-01-12  16-01-12  100  102  102  101

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

Table 3 Concentrations of the test substance in test medium - final test

Time of sampling [hours] Date of sampling [dd-mm-yy]   Date of analysis 1 [dd-mm-yy]         Concentration Relative to nominal [%]   Relative to initial [%]  
Nominal [mg/l] Analysed [mg/l]
 06-02-12  10-02-12  0 / 100  n.d. / 100  n.a. / 100  
 48  08-02-12  10-02-12 0 / 100  n.d. / 100  n.a. / 100 n.a. / 100 

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

n.d. Not detected.

n.a. Not applicable.

3. Combined limit/range-finding test

Immobility ranged between 0 and 20% in the different concentrations (0 - 100 mg TDI-Urone/L) without a relation to the concentration. High numbers of trapped daphnia were observed throughout the test in all solutions. It was expected that the EC50 exceeded the highest concentration tested, but due to the high numbers of floaters it was decided to confirm this in a subsequent limit test. Analytical results of samples taken from nominally 100 mg/l showed that the measured concentration was stable and in agreement with nominal (101 -102%).

4. Limit test - Immobility

The responses recorded in this test confirmed that the EC50 exceeded 100 mg/l. No effects were observed at this exposure concentration throughout the test period. The 5% immobility (1 daphnid) found in the control is within the acceptability criterion for the control.

Table 4 Acute immobilisation of daphnids after 24 and 48 hours in the limit test

Concentration TDI-Urone (mg/l)    Vessel number    Number Daphnia exposed    Response at 24 h      Response at 48 h    
 number  Total %  number  Total %
 Control  A / B / C / D  5 / 5 / 5 / 5 0 / 0 / 0 / 0    0 0 /0 /0 / 1   5
100  A / B / C / D  5 / 5 / 5 / 5 0 / 0 / 0 / 0  0  0 / 0 / 0 / 0  0
Validity criteria fulfilled:
yes
Remarks:
< 10 % immobilsation in controls; oxygen content in control and test vessels was > 3 mg/L.
Conclusions:
TDI-Urone did not induce acute immobilisation of Daphnia magna at 100 mg/l after 48 hours of exposure (NOEC).
The 48h-EC 50 exceeded 100 mg/l based on analytically confirmed nominal concentrations.
Executive summary:

Acute Toxicity Study in Daphnia magna with TDI-Urone.

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 2008 and the ISO International Standard 6341, 1996.

The batch of TDI-Urone tested was a clear colourless powder with a purity of 97.5% active content (being a mixture of isomers) and completely soluble in test medium at the concentrations tested.

A limit test was performed based on the results obtained in a preceding combined limit/range-finding test. Twenty daphnia per test group (4 replicates, 5 per replicate) were exposed to both a control and a TDI-Urone concentration of 100 mg/l. The total test period was 48 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test period.

Analysis of the samples taken from 100 mg/l at the start and the end of the test showed that the measured concentration was stable and in agreement with nominal (100%).

The study met the acceptability criteria prescribed by the protocol and was considered valid. TDI-Urone did not induce acute immobilisation of Daphnia magna at 100 mg/l after 48 hours of exposure (NOEC).

The 48h-EC 50 exceeded 100 mg/l based on analytically confirmed nominal concentrations.

Description of key information

TDI-Urone did not induce acute immobilisation of Daphnia magna at 100 mg/l after 48 hours of exposure (NOEC).
The 48h-EC 50 exceeded 100 mg/l based on analytically confirmed nominal concentrations.

Key value for chemical safety assessment

Additional information

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 2008 and the ISO International Standard 6341, 1996.

The batch of TDI-Urone tested was a clear colourless powder with a purity of 97.5% active content (being a mixture of isomers) and completely soluble in test medium at the concentrations tested.

A limit test was performed based on the results obtained in a preceding combined limit/range-finding test. Twenty daphnia per test group (4 replicates, 5 daphnia per replicate) were exposed to both a control and a TDI-Urone concentration of 100 mg/l. The total test period was 48 hours. Samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test period.

Analysis of the samples taken from 100 mg/l at the start and the end of the test showed that the measured concentration was stable and in agreement with nominal (100%).

The study met the acceptability criteria prescribed by the protocol and was considered valid. TDI-Urone did not induce acute immobilisation of Daphnia magna at 100 mg/l after 48 hours of exposure (NOEC).

The 48h-EC 50 exceeded 100 mg/l based on analytically confirmed nominal concentrations.

In addition, the Degussa 2005 Safety Data Sheet reports the EC50 of TDI-Urone for Daphnia magna to be 1000 mg/L. This information, however, is not considered to be reliable as not details on test design and results are available for assessment.