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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:
16 January 2018 - 25 March 2018
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the freshly prepared control and 100 mg/L loading rate WAF test group from the bulk test preparation at 0 and 24 hours and from the corresponding old or expired pooled replicates 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
Vehicle:
not specified
Details on test solutions:
Reconstituted water (Elendt M7) used for both the range finding and definitive test.
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 WAF of the test item.

Preliminary work was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.
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 2) 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:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
18 to 22 °C
pH:
8.1
Dissolved oxygen:
8.3
Details on test conditions:
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 (500 mg) was added to the surface of 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. Microscopic observations made on the WAF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 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 to 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.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24 and 48 hours

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.

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.


The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24 Hour old test preparations. 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.
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.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Details on results:
Chemical analysis of the fresh test preparations at 0 and 24 hours (see Annex 5) showed that measured test concentrations of 0.0028 and 0.0040 mg/L were obtained respectively. Chemical analysis of the aged test preparation at 24 and 48 hours showed that measured concentrations of less than the LOQ of the analytical method employed were obtained, determined to be 0.0010 mg/L.
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.

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. A single immobilized daphnia was observed in the 100 mg/L loading rate test concentration, however, as no more than 10% immobilization occurred, this was considered not to have had an impact on the results of the test.
There was no significant immobilization in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time
(Hour) EL50
(mg/L Loading Rate WAF)
24 >100
48 >100
The NOEL after 24 and 48 hours exposure was 100 mg/L loading rate WAF.
It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

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 100 mg/L loading rate was observed to be a clear, colorless water column with globules floating on water surface. After 23 hours stirring and a 1 Hour standing period the 100 mg/L loading rate was observed to remain a clear, colorless water column with oily globules/patches floating on water surface. Microscopic examination of the WAF showed test item observed and therefore it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all of the dispersed test item. During the test the control and 100 mg/L loading rate were observed to be clear, colorless solutions.

Table2            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

5N

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

0

0

0

0

0

0

5 N

5 N

5 N

5 N

100

0

0

1

0

1

5

5 N

5 N

4 N

5 N


R   = Replicate

N    = Normal

Validity criteria fulfilled:
yes
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 NOEL rate was 100 mg/L loading rate WAF.
Executive summary:

Introduction

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, 20 daphnids (4 replicates of 5 animals) were exposed to a WAF of the test item at a nominal loading rate of100 mg/L for 48 hours at a temperature of 20 °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 fresh test preparations at 0 and 24 hours showed that measured test concentrations of 0.0028 and 0.0040 mg/L were obtained respectively. Chemical analysis of the aged test preparations at 24 and 48 hours showed that measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method employed were obtained, determined to be 0.0010 mg/L.

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 (NOEL) 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

The acute toxicity of the test item to the freshwater invertebrateDaphnia magnahas been investigated and gave a 48‑Hour EL50value of greater than 100 mg/L loading rate WAF. The NOEL rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Additional information