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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:
Experimental Starting Date: August 19, 2018 and Experimental Completion Date: September 26, 2018
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
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 17083101
- Expiration date of the lot/batch: August 30, 2019
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, at 20 ± 5 °C. Keep container tightly closed in a dry and well-ventilated place.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100 mg/L (limit test)
- Sampling method: For the determination of the actual test item concentration, duplicate samples were taken at the start and at the end of the test (after 48 hours) from the single test concentration of 100 mg/L and from the control. For the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling. The concentration of HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy) phenoxy]ethanol) was analyzed in both duplicate test medium samples from the single test concentration of 100 mg/L and in one of the duplicate test medium samples from the control from both sampling times (0 and 48 hours).
- Sample storage conditions before analysis: Immediately after sampling, all samples were frozen (at -20 ± 5 °C). In pre-experiments for investigation of the storage stability of the samples, the test item was found to be stable under these storage conditions.
Vehicle:
no
Details on test solutions:
- Method: The test medium of the nominal concentration of 100 mg/L was prepared by dissolving 100.0 mg of the test item completely in 1000 mL of test water. The test item was dissolved using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. After the dissolving procedure the test concentration of 100 mg/L was a clear solution. The test medium was prepared just before the introduction of the daphnids (i.e., start of the test).
This highest concentration of 100 mg/L was used to prepare the test media of the lower concentrations in a series of dilutions. Test procedure and conditions were comparable to those applied in the main test with the following exception: For each treatment, 10 daphnids were randomly distributed into two replicates of five daphnids each.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water-flea
- Strain/clone: Straus
- Justification for species other than prescribed by test guideline:
- Source: Daphnia Collection of the University of Basel / Switzerland in 2015), successfully bred in IES Ltd Laboratories under temperature and light conditions identical to those of the tests.
- Age of parental stock (mean and range, SD): At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.
- Feeding during test: no


ACCLIMATION
- Acclimation period: not indicated
- Acclimation conditions (same as test or not): yes
- Type and amount of food: algal suspension of the green algae Desmodesmus subspicatus supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-GmbH, 49324 Melle / Germany).
- Feeding frequency: three times a week
- Health during acclimation (any mortality observed): no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Remarks on exposure duration:
no
Post exposure observation period:
not applicable
Hardness:
Water hardness: 2.5 mmol/L (250 mg/L as CaCo3)
Test temperature:
21°c
pH:
7.8 (start of the test) and 7.9 (end of the test)
Dissolved oxygen:
8.6 mg/L (start of the test) and 8.2 (end of the test)
Salinity:
n/a
Conductivity:
n/a
Nominal and measured concentrations:
- Range-finding test:
Nominal concentrations: 1.0*, 10 and 100 mg/L
Measured concentrations at t=0h: 9.85, 96 mg/L
Measured concentrations at t=48h: 9.75, 97.0 mg/L

* 1 mg/L not measured

- Final test:
Nominal concentrations: 100 mg/L
Measured concentrations at t=0h: 97.3 mg/L
Measured concentrations at t=48h: 95.5 mg/L

Based on these results the main test was performed as limit test in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to the nominal concentration of 100 mg/L. A control was tested in parallel (test water without addition of the test item).
Details on test conditions:
TEST SYSTEM
- Vessel: 100 mL glass beakers filled with 50 mL of test medium. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.
- Type (delete if not applicable): closed (covered with glass plates to reduce the loss of water by evaporation and to avoid entry of dust into the solutions
- Material, size, headspace, fill volume: 50 ml of test medium (10 mL for each Daphnids)
- Type of flow-through: static test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates

TEST MEDIUM / WATER PARAMETERS
Reconstituted test water (ISO Test water) according to OECD Guideline No. 202 was used in the study. It consisted of analytical grade salts dissolved in purified water at the following nominal concentrations:

Ingredients / Properties Concentration
[mmol/L] [mg/L]
CaCl2 × 2H2O 2.0 294
MgSO4 × 7H2O 0.5 123
NaHCO3 0.75 65
KCl 0.075 5.8
Water Hardness 2.5 250 as CaCO3
Alkalinity 0.8 ---

The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The test water was aerated prior to the start of the study until oxygen saturation was reached.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used.
- Light intensity: Light intensity during the light period was between 15 and 17 µmol m-2 s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilization / pH / Oxygen dissolved / Temperature

VEHICLE CONTROL PERFORMED: no vehicle was used in the test

RANGE-FINDING STUDY
- Test concentrations: 1.0 / 10 / and 100 mg/L
- Results used to determine the conditions for the definitive study: as immobilisation was seen in the range finding test at all three concentrations, 100 mg/L was selected as the concentration to be used in the main test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
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
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
* no remarkable observations were made concerning the appearance of the test medium. The test medium was clear solution throughout the entire test duration.
* In the control and in the test item concentration of 100 mg/L, no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration or unusual behavior such as trapping at the surface water) during the test period of 48 hours

* 48-h NOEC: >= 100 mg/L
* 48-h EC100: > 100 mg/L


Results with reference substance (positive control):
- Results with reference substance valid? yes
- ECx: result of the latest positive control test in April 2018 (24-hour EC50: 1.1 mg/L) showed that the sensitivity of the test organisms was within the range given by the guideline (24-hour EC50: 0.60 2.1 mg/L).
Reported statistics and error estimates:
No EC50 could be calculated because the test item proved to be non-toxic (EC50 > highest concentration tested)

Effect of HER on the Mobility of Daphnia magna

Nominal
Test Item
Concentration

No. of
Daphnids
Tested

Immobilized
Daphnids after
24 Hours

Immobilized
Daphnids after
48 Hours

[mg/L]

 

No.

[%]

No.

[%]

Control

20

0

0

0

0

100

20

0

0

0

0

pH Values, Dissolved Oxygen Concentrations and Temperature in the Treatments

Nominal
Test Item
Concentration

Start (0 Hours)

End (48 Hours)

[mg/L]

pH

Oxygen
[mg/L]

Temperature
[°C]

pH

Oxygen
[mg/L]

Temperature
[°C]

Control

7.9

8.9

21

7.8

8.8

21

100

7.9

8.6

21

7.8

8.2

21

Validity

The test is considered to be valid, as in the control no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration or unusual behavior such as trapping at the surface water). Furthermore, the dissolved oxygen concentration at the end of the test was 3 mg/L in the control and test vessels.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item on Daphnia magna was determined in a 48-hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2. A limit test was performed in accordance to demonstrate that the test item has no toxic effect on the test organisms up to and including a nominal concentration of 100 mg/L. Thus, the only nominal concentration tested was 100 mg/L. A control group was tested in parallel.  The test item had no acute toxic effects on Daphnia magna in a static 48 hour test up to the nominal concentration of 100 mg/L under the conditions of the test.
Executive summary:

The acute toxicity of the test item on Daphnia magnawas determined in a 48‑hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2.

 

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test organisms up to and including a nominal concentration of 100 mg/L. Thus, the only nominal concentration tested was 100 mg/L. A control group was tested in parallel.

 

The measured concentration of the test item in the test medium of the single test concentration of 100 mg/L was 97 and 95 % of nominal value at the start and at the end of the test, respectively.

 

Thus, the correct dosage of the test item was confirmed. The test item was stable in the test medium over the test period of 48 hours. Therefore, the endpoint values were related on the nominal concentration of 100 mg/L.

 

The biological test results (based on nominal concentration) were as follows:

 

 EC values

Immobility

 Nominal Test Item concentration

(mg/L)

24-hour EC50

> 100

24-hour EC0

≥100

24-hour EC100

> 100

48-hour EC50

  

> 100

48-hour EC0

≥100

48-hour NOEC

≥100

48-hour EC100

> 100

In conclusion, the test item had no acute toxic effects on Daphnia magna in a 48-hour static test up to the nominal concentration of 100 mg/L.

Description of key information

The acute toxicity of the test item on Daphnia magna was determined in a 48-hour static test according to the OECD Guideline for Testing of Chemicals, No. 202 (2004) and the Commission Regulation (EC) No. 440/2008, Part C.2. A limit test was performed in accordance to demonstrate that the test item has no toxic effect on the test organisms up to and including a nominal concentration of 100 mg/L. Thus, the only nominal concentration tested was 100 mg/L. A control group was tested in parallel.  The test item had no acute toxic effects on Daphnia magna in a static 48 hour test up to the nominal concentration of 100 mg/L under the conditions of the test.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information

The test item is not toxic for invertebrate.