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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:
25 Jul - 29 Sep 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Federal Office of Public Health, Bern, Switzerland

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, and limit concentration 100 mg/L (nominal)
- Sampling method: For the determination of the actual test item concentration, duplicate samples were taken at the start and end of each test medium renewal periods from the test concentration and from the control. For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling. Immediately after sampling, methanol (10 mL methanol per 100 mL sample volume) was added to each sample to stabilize the latter during the storage period. The concentration of the test item 10-HSA was analytically measured in the duplicate test medium samples of the single test concentration taken at the start and end of each test medium renewal period. From the control, one of the duplicate samples was analyzed per sampling time.
- Sample storage conditions before analysis: All samples were deep-frozen (at about -20 °C). Based on pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Due to the low water solubility of the test item, a suspension with the loading rate of 100 mg/L was prepared at the start of the test and before each test medium renewal by dispersing 100 mg of the test item (dosing range: 100.02 and 100.05 mg) in 1000 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer for three hours at room temperature in the dark to dissolve a maximum amount of the test item in the test water. After stirring, the suspension of the test item was filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 µm). As a pre-caution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. The undiluted filtrate with a loading rate of 100 mg/L was used as test medium. The test medium was prepared just before the introduction of the daphnids (i.e., start of the test) and prior to the test medium renewal after 24 hours.
- Controls: yes, test water without test item.
- Evidence of undissolved material: The test medium was a clar solution throughout the entire test duration.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: originally from the Daphnia Collection of the University of Basel, Switzerland (2015)
- Source: in-house culture at IES Ltd Laboratories
- Age of daphnia at the start of the test: 6 - 24 h old and not first brood progeny
- Feeding during test: no

BREEDING:
- Food type: algal suspension of the green algae Desmodesmus subspicatus, strain n° 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, Göttingen, Germany) and cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal supension and a commercial fish diet (Tetra Min Hauptfutter, TETRA-Werke, Melle, Germany)
- Frequency: three times a week

ACCLIMATION
- No, the daphnia were bred under temperature and light conditions identical to those of the tests.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
2.5 mmol/L = 250 mg/L as CaCO3 (reconstituted test water)
Test temperature:
21 °C
pH:
7.6 - 7.8 (control)
7.8 - 7.9 (treatment)
Dissolved oxygen:
8.4 - 8.7 mg/L (control)
8.1 - 8.5 mg/L (treatment)
Nominal and measured concentrations:
control, 100 mg/L (nominal)
< LOQ, 0.26 mg/L (arithmetic mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: beakers
- Type: The beakers were covered with transparent, disposable polypropylene sheets to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
- Material, size, fill volume: Material: glass; Size: 100 mL; Fill volume: 50 mL
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached.
- Renewal rate of test solution: after 24 h (one time during entire test)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water, consisting of analytical grade salts dissolved in purified water according to ISO 6341
- Alkalinity: 0.8 mmol/L
- Ca/mg ratio: 4:1 (based on molarity)
- Na:K ratio: 10:1 (based on molarity)
- Culture medium different from test medium: no
- Intervals of water quality measurement: At the start and end of each test medium renewal period, the pH values, dissolved oxygen concentration and water temperature were determined in each treatment. In addition, the appearance of the test medium was visually controlled and documented.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 h dark
- Light intensity: 15 - 18 µmol * m -² * s-¹

EFFECT PARAMETERS MEASURED:
- Immobilisation of daphnia: visual inspection after 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: A stirring pre-experiment (GLP) with three individual suspensions of 100 mg/L test item (nominal) with different stirring regimes (3, 24, and 96 h) was performed to determine the solubility of the poorly soluble test item (water solubility < 100 mg/L) in test water and to determine the optimum time period to reach equilibration within a reasonable time period.
- Results used to determine the conditions for the definitive study: The analytically determined concentration of dissolved test item in the filtrates were 0.36 mg/L (3 h), 0.25 mg/L (24 h), and 0.26 mg/L (96 h). These results showed that the maximum concentration of dissolved test item in test water was reached after a stirring period of 3 h.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrations
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.26 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: 0%
- Effect concentrations exceeding solubility of substance in test medium: yes, the analytically measured concentration of 0.26 mg/L corresponds to the solubility limit of the test item in test water at a loading rate of 100 mg/L (nominal).
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (24 h) = 1.6 mg/L potassium dichromate (Mar 2016)
Reported statistics and error estimates:
The 24-hour and 48-hour EC/EL-values could not be calculated due to the absence of a toxic effect of the test item. Therefore, the EC0/EL0, EC50/EL50, EC100/EL100 and the NOEC/NOEL were directly determined from the raw data.

Any other information on results incl. tables

RESULTS OF EXPERIMENTAL CONDITIONS

Table 1. pH Values in the Treatments.

Mean measured
test item
concentration

Exposure time

 

 

 

0 h

24 h

48 h

[mg/L]

new

old

new

old

Control

7.8

7.9

7.9

7.8

0.26

7.6

7.8

7.7

7.8

Table 2. Dissolved Oxygen Concentrations [mg/L] in the Treatments.

Mean measured
test item
concentration

Exposure time

 

 

 

0 h

24 h

48 h

[mg/L]

new

old

new

old

Control

8.7

8.5

8.6

8.4

0.26

8.1

8.3

8.2

8.5

ANALYTICAL RESULTS

The test item concentration remained stable during the test (Table 3).

 

Table 3. Results for Test Samples

Sampling Day /
Sample Age

Loading Rate of 100 mg Test Item/L

Measured Concentration
 of Test Item x
 

Sample Preparation Factor
F

Determined Concentration of Test Item
c

Sample 1

Sample 2

Mean Value

 

[day/hours]

 

[mg/L]

[mg/L]

[mg/L]

 

[mg/L]

0/0

Control

n.d.

n.d.

<LOQ

0.0500

n.a.

(fresh)

Undiluted Filtrate

2.62

2.47

2.54

0.100

0.254

1/24

Control

n.d.

n.d.

<LOQ

0.0500

n.a.

(aged)

Undiluted filtrate

2.41

2.89

2.65

0.100

0.265

1/0

Control

n.d.

n.d.

<LOQ

0.0500

n.a.

(fresh)

Undiluted Filtrate

2.60

2.46

2.53

0.100

0.253

2/24

Control

n.d.

n.d.

<LOQ

0.0500

n.a.

(aged)

Undiluted Filtrate

2.72

2.48

2.60

0.100

0.260

LOQ      = 0.0145 mg test item /L

n.d.        = not detected

n.a.        = not applicable

No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solutions throughout the entire test duration.

The biological results were related to the mean measured test item concentration of 0.26 mg/L and to the loading rate of 100 mg/L.

BIOLOGICAL RESULTS

During the test period of 48 h, no immobilized test organisms were determined in the control and the limt test concentration of 0.26 mg/L (loading rate of 100 mg/L).

Table 4. Effect of test item on the Mobility of Daphnia magna.

Mean measured
test item
concentration

No. of
daphnids
tested

Immobilized
daphnids after
24 hours

Immobilized
daphnids after
24 hours

Immobilized
daphnids after
48 hours

Immobilized
daphnids after
48 hours

[mg/L]

 

No.

[%]

No.

[%]

Control

20

0

0

0

0

0.26*

20

0

0

0

0

 *: Undiluted filtrate of a suspension with a loading rate of 100 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test item 10-HSA had no acute toxic effects on Daphnia magna in a 48-hour semi-static test up to the analytically measured concentration of 0.26 mg/L which corresponds to the solubility limit of the test item in test water at a loading rate of 100 mg/L.
Executive summary:

The acute toxicity of the test item10-HSA to 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.

 

Due to the low water solubility of the test item, a suspension of the test item with the loading rate of 100 mg/L was prepared by using ultrasonic treatment for 15 minutes and intensive stirring for 3 hours to reach a maximum concentration of dissolved test item in test water. The stirring time was based on a pre-experiment. After stirring, the suspension was filtered through a 0.45 µm membrane filter. The undiluted filtrate was used as test medium.

 

The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

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 the loading rate of 100 mg/L. Thus, the only loading rate tested was 100 mg/L. Additionally a control was tested in parallel.

 

The measured concentration of 10-HSA in the test medium with a loading rate of 100 mg/L was 0.25 mg/L at the start of both renewal periods. At the end of the two 24-hour renewal periods, 103 and 104 % of the initially measured concentration were found. Thus, the test item was sufficiently stable during the test medium renewal periods of 24 hours. The mean measured concentration during the test period of 48 hours was calculated to be 0.26 mg/L (arithmetic mean over all measurements of test concentration).

 

The biological results were related to the mean measured test item concentration of 0.26 mg/L and to the loading rate of 100 mg/L:

 

24-hour EC50/EL50         > 0.26/ >100 mg/L

  24-hour EC0/EL0        0.26 /100 mg/L

  24-hour EC100/EL100        > 0.26 / > 100 mg/L

  48-hour EC50/EL50        > 0.26 / > 100 mg/L

  48-hour EC0/EL0 and  48-hour NOEC/NOEL        0.26 / 100 mg/L

  48-hour EC100/EL100        > 0.26 / > 100 mg/L

 

In conclusion, the test item 10-HSA had no acute toxic effects on Daphnia magna in a 48-hour semi-static test up to the analytically measured concentration of 0.26 mg/L which corresponds to the solubility limit of the test item in test water at a loading rate of 100 mg/L.