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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:
01 May 2018 to 28 Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
yes
Remarks:
During the combined limit/range-finding test, temperature was not constant within ± 1°C as indicated by the study plan, however since temperature deviated for only a short time, this has no effect on the study.
Principles of method if other than guideline:
Test was conducted according to a standard OECD guideline.
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Thio Isocyanate Adduct
Batch number: BZA13264 and BZA15779
Expiry date: 23 June 2018 and 23 August 2018
Test item storage: At room temperature under nitrogen
Appearance: Colourless liquid
Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken from all test concentrations and the control at the start of the test and after 24 hours from the freshly prepared solutions and at the first renewal (24 hours) and at the end of the test from the 24 hour old solutions. Sample volumes of 2.0 mL where taken from the approximate centre of the test vessels and were subsequently analysed on the day of sampling (i.e. samples were not stored for later analysis). At the end of each refreshment period, the replicates were pooled at each concentration before sampling.
Vehicle:
no
Details on test solutions:
Preparation of test solutions used in the combined limit/range-finding test started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A two day period of magnetic stirring was applied to accelerate dissolution of the test item in medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.

Preparation of test solutions used in the final test started with loading rates individually prepared at 10, 18, 32, 56 and 100 mg/L. A three hour period of magnetic stirring was applied to accelerate dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of 15 minutes. Thereafter, the aqueous WAFs were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.

Any residual volumes were discarded.
Test organisms (species):
Daphnia magna
Details on test organisms:
Source: In-house laboratory culture with a known history
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.
Characteristics: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
Standard duration
Post exposure observation period:
None, not a standard test requirement
Hardness:
180 mg/L CaCO3
Test temperature:
20°C
pH:
7.5 to 8.1
Dissolved oxygen:
8.3 to 9.0 mgO2/L
Salinity:
NA
Conductivity:
NA
Nominal and measured concentrations:
WAFs prepared at loading rates of: 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
Combined Limit/range-finding test:
Twenty daphnids per concentration (four replicates, five daphnids per vessel) were exposed to a control and a WAF prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the exception that ten daphnids per concentration (5 per vessel) were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L in the combined range finding test, solutions were not renewed after 24 hours of exposure, dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration at the end of the test, samples for determining actual exposure concentrations were only taken from the control and the highest test concentration at the start and the end of the test.

Pre-test:
A pre-test was conducted to determine the optimum stirring time to dissolve the test item in test medium for the final test, in which WAFs were prepared at loading rates of 1.0 and 100 mg/L and a 48, 24, 6, 3 and 1 hour period of magnetic stirring was applied, followed by a settling period of 15 minutes. Samples were taken for analysis to determine actual concentrations after the settling period. Based on these results, the optimum stirring period was determined (3 hours) for the final test.

Final test:
Twenty daphnids per concentration (five per vessel containing 80 ml of test solution) were exposed to a control and the five prepared test concentration WAFs for 48 hours, with renewal of test solutions after 24 hours. Test organisms were not fed during the test. The test was conducted under lighting of 16 hours photoperiod daily and there was no aeration of the test solutions. Immobility (including mortality) of test organisms was measured at 24 and 48 hours. Measurements of pH and dissolved oxygen were taken at the beginning, after 24 hours of exposure and at the end of the test, for all concentrations and the control. Temperature of test medium was measured continuously in a temperature control vessel, beginning at the start of the test and also after 24 hours of exposure from the old and renewed test solutions.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: Loading rate
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: Loading rate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 0.006 mg/L
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: Adduct, first constituent
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.006 mg/L
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: Adduct, first constituent
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 5.1 mg/L
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: Adduct, second constituent
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 5.1 mg/L
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: Adduct, second constituent
Details on results:
Combined limit/range-finding Test:
No immobility was observed in the control and at any of the test concentrations throughout the test. Therefore, the expected EC50 was above a WAF prepared at a loading rate of 100 mg/L.

Final test:
No immobility was observed in the control and at any of the test concentrations throughout the test.
Results with reference substance (positive control):
The positive control used potassium dichromate as the reference item at nominal concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L and to a control. Twenty daphnids were exposed per concentration. The 48 hour EC50 was 0.59 mg/L (with a 95% confidence interval between 0.53 and 0.72 mg/L). The results from the positive control were 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, thus the sensitivity of this batch of D. magna was in agreement with the historical data collected at the testing laboratory.
Reported statistics and error estimates:
No EC50 could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested).

Immobility in the final test

Time (h)

Replicate

Thio Isocyanate Adduct Loading Rate (mg/L)

Control

10

18

32

56

100

0

A

5

5

5

5

5

5

B

5

5

5

5

5

5

C

5

5

5

5

5

5

D

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

24

A

0

0

0

0

0

0

B

0

0

0

0

0

0

C

0

0

0

0

0

0

D

0

0

0

0

0

0

Total immobilised

0

0

0

0

0

0

Effect %

0

0

0

0

0

0

48

A

0

0

0

0

0

0

B

0

0

0

0

0

0

C

0

0

0

0

0

0

D

0

0

0

0

0

0

Total immobilised

0

0

0

0

0

0

Effect %

0

0

0

0

0

0

Validity criteria fulfilled:
yes
Remarks:
None of the control daphnids showed immobilisation or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.
Conclusions:
The 48 h-EC50 for Daphnia magna exposed to Thio Isocyanate Adduct was beyond the range of concentrations tested, i.e. exceeded an average exposure concentration of 0.0062 mg/L for the first constituent and of 5.1 mg/L for the second constituent being considered the maximum soluble concentration of the test item in test medium.
Executive summary:

The short-term toxicity of Thio Isocyanate Adduct to the freshwater aquatic invertebrate, Daphnia magna, was assessed via an OECD Guideline 202 (‘Daphnia sp. Acute Immobilisation Test’) study. The study was performed at a GLP accredited laboratory, fulfilled the validity criteria described by the guideline and is well documented and is therefore considered to be reliable.  

                                                                                                                  

Exposures of D. magna to the test item showed that the 48 hour EC50 was beyond theconsidered maximum soluble concentration of the test item(i.e.exceeded an average exposure concentration of 0.0062 mg/L for the first constituent and of 5.1 mg/L for the second constituent).

Description of key information

The short-term toxicity of Thio Isocyanate Adduct to the freshwater aquatic invertebrate, Daphnia magna, was assessed via an OECD Guideline 202 (‘Daphnia sp. Acute Immobilisation Test’) study. The study was performed at a GLP accredited laboratory, fulfilled the validity criteria described by the guideline and is well documented and is therefore considered to be reliable.  

                                                                                                                  

Exposures of D. magna to the test item showed that the 48 hour EC50 was beyond theconsidered maximum soluble concentration of the test item(i.e.exceeded an average exposure concentration of 0.0062 mg/L for the first constituent and of 5.1 mg/L for the second constituent).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information