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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:
This study was conducted between 24 July 2017 and 03 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study conducted according to OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
EC No 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification: 4,4’-(1,3-Phenylenediisopropylidene) diphenylcyanate
Physical state/Appearance: Yellow viscous liquid
Batch: FAR366085A
Purity: Complex mixture
Expiry Date: 29 September 2019
Storage Conditions: ≈ 4 °C in the dark
Analytical monitoring:
yes
Details on sampling:
A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only the concentration to be used for the definitive test was analyzed.
Vehicle:
yes
Remarks:
Reconstituted water (ISO medium)
Details on test solutions:
Range-finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
A nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no immobilization or adverse reactions to exposure were observed.
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity
Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium (see Annex 1) in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Remarks:
Elendt M7 medium
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
18-22 ±1”C
pH:
The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC
Dissolved oxygen:
equal to or greater than 3 mg/L
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item
Details on test conditions:
Experimental Design and Study Conduct

Validation of Mixing Period
Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF

Range-finding Test
The loading rates to be used in the definitive test were determined by a preliminary range-finding test.
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added to a glass slide and suspended in the water column of 5 liters of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.
In the range-finding test 5 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only the concentration to be used for the definitive test was analyzed.

Definitive Test
Based on the results of the range-finding test a "limit test" was conducted at a loading rate of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines no immobilization or adverse reactions to exposure were observed.
A nominal amount of test item (250 mg) was added to a glass slide and suspended in the water column of 2.5 liters of test water to give the 100 mg/L loading rate. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after
23 hours and the mixture allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF during the initial preparation showed no micro-dispersions or undissolved test item to be present. During the second preparation, visual observation of the water column at the end of the stirring period showed that undissolved test item was dispersed throughout, and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed that no undissolved test item remained in the media.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 and 48 hours.

Exposure Conditions
As in the range-finding test 150 mL glass jars containing approximately 100 mL of test preparation were used. At the start of the test five daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test item.
Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media.


Assessments

Test Organism Observations
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Water Quality Criteria
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period.

Chemical Analysis of Test Loading Rates
Samples were taken from the control and the 100 mg/L loading rate WAF test group from the bulk test preparation at 0 and 24 hours and from the pooled replicates (replicates R1 – R4) at 24 and 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described above.

Data Evaluation
Statistical Analysis
An estimate of the EL50 values was given by inspection of the immobilization data.

Validation Criteria
The results of the test are considered valid if the following performance criteria are met:
• No more than 10% of the control daphnids show immobilization or other signs of disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface water).
• The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.

Reference substance (positive control):
yes
Remarks:
Potassiium dichromate
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
Range-finding Test
No immobilization was observed at 1.0, 10 and 100 mg/L loading rate WAF.
No sub-lethal effects of exposure were observed throughout the test.
Based on this information, a single loading rate of four replicates, of 100 mg/L, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.
Chemical analysis of the 100 mg/L loading rate test preparations at 0 hours (see Annex 4) showed measured that a measured test concentration of 3.7 mg/L was obtained, and at 48 hours showed that a measured concentration of 0.28 mg/L was obtained. This indicated that the test item was not stable under test conditions.

Definitive Test
Chemical Analysis of Test Loading Rates
Chemical analysis of the freshly prepared test preparations at 0 and 24 hours (see Annex 4) showed measured test concentrations of less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.0053 mg/L and 0.018 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to be less than the LOQ.
The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Immobilization Data
Cumulative immobilization data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2.
There was no immobilization in 20 daphnids exposed to a test concentration of 100 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time (h) EC50 (mg/L)
24 >100
48 >100

The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Sub-Lethal Effects
No sub-lethal effects of exposure were observed throughout the test.

Validation Criteria
The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization 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

Water Quality Criteria
Temperature was maintained at approximately 21 - 22”C throughout the test. While there were no treatment related differences for oxygen concentration, concentration dependent differences in pH were observed at 0-Hours.
Throughout the test the light intensity was observed to be in the range 677 to 800 Lux.


Vortex Depth Measurements
The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

Observations on Test Item Solubility
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the first and second mixing periods the 100 mg/L loading rate was observed to be a clear colorless water column with test item adhered to a glass slide suspended in the water column. After 23 hours stirring and a 1-Hour standing period during the first preparation, the 100 mg/L loading rate was observed to be a clear colourless water column with test item adhered to a microscope slide and at the base of the vessel. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After 23 hours stirring and a 1-Hour standing period during the second preparation, the
100 mg/L loading rate was observed to be a clear colorless water column with test item adhered to a microscope slide, at the base of the vessel and dispersed throughout. Examination of the WAF showed dispersed material throughout the water column and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all of the dispersed material.
At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Results with reference substance (positive control):
A positive control (Envigo Study Number XT01HS) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
Exposure conditions for the positive control were similar to those in the definitive test, however, throughout the positive control the temperature range was recorded between 19 and 22 °C, therefore outside of the range quoted in the study plan of 18 to 22 °C with a maximum deviation of ±1 °C during the test. This deviation was considered not to have adversely affected the results of the test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

Time Point (Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
24 1.3 1.0-1.8 1.0 1.8
48 1.2 1.1-1.3 0.56 1.0
The No Observed Effect Concentration is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item

Table 2: Cumulative Immobilization Data and Observations in the Definitive Test

Nominal
Loading Rate
(mg/L)

24 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

Nominal
Loading Rate
(mg/L)

48 Hours

Cumulative Immobilized Daphnia
(Initial Population: 5 Per Replicate)

Observations

R1

R2

R3

R4

Total

%

R1

R2

R3

R4

Control

1

0

0

0

0

0

4 N

5 N

5 N

5 N

100

0

0

0

0

0

0

5 N

5 N

5 N

5 N

R1 – R4 = Replicates 1 to 4

N = Normal

Validity criteria fulfilled:
yes
Remarks:
Considered to be valid given that none of the control daphnids showed immobilization 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 acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF.
Executive summary:

A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 Methods

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under semi-static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results…….

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours showed measured test concentrations of less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.0053 mg/L and 0.018 mg/L. Chemical analysis of the aged test preparations at 24 and 48 hours showed measured test concentrations to be less than the LOQ.

The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Exposure ofDaphnia magnato the test item gave EL50values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Description of key information

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information