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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:
The study was conducted between 24 November 2016 and 27 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Identification: X-19574
Nature of Test Item: UVCB
Physical state/Appearance: Amber colored viscous liquid
Batch: X-19574-00-00
Purity: Not applicable (100% as UVCB)
Expiry Date: 01 June 2017
Storage Conditions: Room temperature in darkness

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Range-finding test:
A sample of each loading rate Water Accomodated Fraction (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 concentrations within the range to be used for the definitive test were analyzed.

Definitive test:
Samples were taken from the control and each loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates (replicates R1 – R4) at 48 hours for quantitative analysis. Samples were stored frozen prior to analysis.
Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
Range-finding test:
Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added to the surface 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 95 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.
Due to high immobilization in the initial range-finding test, in the second range-finding test, Daphnia magna were exposed to a series of nominal loading rates of 0.0010, 0.010, 0.10 and 1.0 mg/L.
A nominal amount of test item (20 mg) was added to the surface of 20 liters of test water to give the 1.0 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 95 hours and the mixtures allowed to stand for 2 hours. 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 mg/L loading rate WAF. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give the 0.10, 0.010 and 0.0010 mg/L loading rate WAFs. Due to the low test concentration, it was not practical to prepare the test concentrations as individual WAFs and dilutions were required.

Definitive test:
A nominal amount of test item (20 mg) was added to the surface of 20 liters of test water to give the 1.0 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 95 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 mg/L loading rate WAF. A series of dilutions was made from the 1.0 mg/L loading rate WAF to give the 0.10, 0.050, 0.025, 0.0125 and 0.00625 mg/L loading rate WAFs.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium 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.

Study design

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

Test conditions

Test temperature:
20 ºC to 22 ºC
pH:
0 Hours: 8.0 - 8.1
48 Hours: 7.8 - 7.9
Dissolved oxygen:
0 Hours: 8.7 - 9.4 mg O2/L
48 Hours: 7.8 - 7.9 mg O2/L
Nominal and measured concentrations:
Nominal concentrations:
Initial range-finding test: 1.0, 10 and 100 mg/L.
second range-finding test: 0.0010, 0.010, 0.10 and 1.0 mg/L
Definitive test: : 0.00625, 0.0125, 0.025, 0.050 and 0.10 mg/L.

Chemical analysis of the 0.050 and 0.10 mg/L loading rate WAF fresh test preparations at 0 hours showed measured test concentrations of 0.042 and 0.083 mg/L, respectively. Chemical analysis of the 0.10 mg/L loading rate WAF aged test preparation at 48 hours showed a measured test concentration of 0.0050 mg/L. The 0.050 mg/L loading rate had declined to less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.0019 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.
The ion that was monitored by positive mode LC-MS had a m/z of 268. This mass corresponds with the [M+H]+ ion of an unsaturated C18 amine (C18H37N). A reference material was not analyzed to confirm the compound being measured.
The test item concentrations were based on the amount of test item weighed out to prepare the standards. No correction of the test item concentration for the content of the amine component was undertaken. Whilst only the amine component of the test item was measured, the concentration of the standards was in terms of test item (mg/L), as such the calculated values for the test solutions are in the same term (mg/L test item).
As the test item is UVCB in nature, the dissolved portion 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.
Details on test conditions:
Throughout the test the light intensity was observed to be in the range 593 to 858 lux.

Range-finding test:
In the range-finding tests, 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 °C 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. Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours, the number of immobilized daphnids was recorded.
The control group was prepared using the WAF methodology and maintained under identical conditions, but not exposed to the test item.

Definitive test:
Exposure Conditions
In the definitive test, 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test, 5 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 °C 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 prepared using the WAF methodology and maintained under identical conditions, but not exposed to the test item.
The test preparations were not renewed during the exposure period.

Test Water
Reconstituted water (ISO medium) was used for the initial range-finding test. Reconstituted water (Elendt M7 medium) was used for the second range-finding and definitive tests.

Reference substance (positive control):
yes
Remarks:
A positive control (Envigo Study Number XT01HS) used potassium dichromate as the reference item. The positive control was conducted between 20 October 2016 and 22 October 2016.

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
0.071 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
geometric mean of the highest loading rate showing no immobilization and the lowest loading rate showing 100% immobilization
Basis for effect:
mobility
Remarks on result:
other: 95% Confidence Limits (mg/L Loading Rate WAF): 0.050 – 0.10
Details on results:
Range-finding Tests
In the initial range-finding test, all daphnia in the 1.0, 10 and 100 mg/L loading rate WAFs were immobilized at 24 hours. The test was therefore terminated at 24 hours.
In the second range-finding test, no immobilization was observed at 0.0010 and 0.010 mg/L loading rate WAFs; however, immobilization was observed at 0.10 and 1.0 mg/L loading rate WAFs.
Based on this information loading rates of 0.00625, 0.0125, 0.025, 0.050 and 0.10 mg/L were selected for the definitive test.
Chemical analysis of the 0.010 and 0.10 mg/L test preparations at 0 hours showed measured test concentrations of 0.036 and 0.11 mg/L, respectively. There was a significant decline in the measured concentrations at 48 hours indicating that the test item was not stable under test conditions.

Definitive Test
Immobilization Data
Analysis of the immobilization data by the geometric mean method at 24 and 48 hours based on the nominal loading rates gave the following results:
Time (h) EL50 95% Confidence limits
(mg/L Loading Rate WAF) (mg/L Loading Rate WAF)
24 0.071 0.050 – 0.10
48 0.071 0.050 – 0.10
The No Observed Effect Loading Rate WAF after 24 and 48 hours exposure was 0.050 mg/L. Correspondingly, the Lowest Effect Loading rate WAF was 0.10 mg/L.

Sub-Lethal Effects
A sub-lethal effect of exposure was observed in the 0.0125 mg/L loading rate WAF. The response was trapping at the surface.

Validation Criteria
The test was 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 greater than 3 mg/L in the control and test vessels.
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.

24 hrs:
EC50 (mg/L) - 1.3 (1.0 - 1.8, 95% confidence limits)
NOEC (mg/L) - 1.0
LOEC (mg/L) - 1.8

48 hrs:
EC50 (mg/L) - 1.2 (1.1 - 1.3, 95% confidence limits)
NOEC (mg/L) - 0.56
LOEC (mg/L) - 1.0

Any other information on results incl. tables

Vortex Depth Measurements

The vortex depths were recorded at the start and end of the mixing period, and were observed to be approximately 1% of the water column height in the control and 1.0 mg/L loading rate.

 Observations on Test Item Solubility

Observations on the test media were carried out during the mixing and testing of the WAF.

At the start of the mixing period, the 1.0 mg/L loading rate was observed to be clear colorless water column with amber oily globules of test item on the surface. After 23 hours stirring and a 1-Hour standing period, it was observed to be a clear colorless water column with particles of test item floating on the surface.  Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After siphoning the 1.0 mg/L loading rate was observed to be a clear, colorless solution. 

At the start and throughout the test, all control and test solutions were observed to be clear, colorless solutions.

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

0.00625

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.0125

0

0

0

0

0

0

5 N

5 N

3 N, 2 T

5 N

0.025

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.050

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.10

5

5

5

5

20

100

A/I

A/I

A/I

A/I

 

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

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.00625

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.0125

0

0

0

0

0

0

5 N

5 N

4 N, 1 T

5 N

0.025

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.050

0

0

0

0

0

0

5 N

5 N

5 N

5 N

0.10

5

5

5

5

20

100

A/I

A/I

A/I

A/I

R1– R4= Replicates 1 to 4

N = No sub-lethal effects observed

T = Trapped at surface

A/I = All daphnia immobilized

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Exposure of the freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results based on nominal loading rates:
Time Point: 48 hrs
EL50 (mg/L Loading Rate WAF): 0.071 95% Confidence Limits (mg/L Loading Rate WAF): 0.050 – 0.10
No Observed Effect Loading Rate (NOEL) (mg/L): 0.050
Lowest Observed Effect Loading Rate (LOEL) (mg/L): 0.10

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., 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 preliminary range-finding tests, twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.00625, 0.0125, 0.025, 0.050 and 0.10 mg/L for 48 hours at a temperature of 20°C to 22 °C under static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Chemical analysis was based on measurement of an unsaturated C18 amine component of the test item. No reference material was analyzed to confirm the compound measured.

Chemical analysis of the 0.050 and 0.10 mg/L loading rate WAF fresh test preparations at 0 hours showed measured test concentrations of 0.042 and 0.083 mg/L, respectively. Chemical analysis of the 0.10 mg/L loading rate WAF aged test preparation at 48 hours showed a measured test concentration of 0.0050 mg/L. The 0.050 mg/L loading rate had declined to less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.0019 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

As the test item is UVCB in nature, the dissolved portion 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 of Daphnia magna to the test item gave the following results:

Time Point

(Hours)

EL50
(mg/L Loading Rate WAF)

95% Confidence Limits (mg/L Loading Rate WAF)

No Observed Effect Loading Rate (NOEL) (mg/L)

Lowest Observed Effect Loading Rate (LOEL) (mg/L)

48

0.071

0.050 – 0.10

0.050

0.10