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Diss Factsheets

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Sampling and analysis

Analytical monitoring:
Details on sampling:
At exposure initiation 50 mL samples of each treatment were collected and at exposure termination 50 mL of pooled treatments were collected.

Test solutions

Details on test solutions:
Due to the result of the preliminary test of the IPO study, code W/30/18 (Pseudokirchneriella subcapitata – growth inhibition test), the test item was considered as not stable during the 48 h preparation period in an open system in the AAP medium. Hence, a solubility test in the closed system in the Elendt M7 medium was performed. The influence of shaking time, settling phase time, temperature and light presence on the solubility of the test item was checked. The solubility test was performed using test item loading rates in the range of 58.7 – 93.6 mg/L. Defined amounts of the test item were weighed separately in glass vessels. Glass vessels were then filled up with Elendt M7 medium to full volume (without headspace) and tightly closed with glass caps. Each test item loading rate was transparent and heterogeneous with undissolved particles.
Additionally four replicates of the control (i.e. test medium) were prepared. Glass vessels were separated into four groups with different pretreatment conditions (i.e. A, B, C, D), each pretreatment group comprising two vessels with defined loading rates of the test item and one replicate each of the control. After 24 h of shaking (A and C), and after 48 h of shaking/settling (B and D), one replicate from each pretreatment group was filtered, and subsequently all replicates were transferred for chemical analysis.
Based on the results of this solubility test, the preliminary test was designed. The pretreatment included 24 h shaking phase (room temperature, darkness, 50 rpm) followed by a 24 h of settling phase (room temperature, darkness).

The definitive test was performed using the filtrate of the test item loading rate of 50 mg/L and the 2-fold, 4-fold, 8-fold and 16-fold diluted filtrate of a loading rate of 50 mg/L plus the control in a static test design.
Appropriate amount of the test item was weighed in a glass crystalliser, transferred into a volumetric flask and filled up to full volume (without headspace) with the Elendt M7 medium. The test item loading rate of 50 mg/L was heterogeneous, with undissolved particles on the surface. The test item loading rate and control were mechanically shaken for 24 h followed by 24 h of settling phase and filtered through a conditioned (pre-filtering a small volume of either the test loading rate or control to saturate the filter) nitrocellulose membrane filter (0.22 Um, GSTF, Millipore). Lower test item concentrations were prepared by serial dilutions of the filtrate of the test item loading rate 50 mg/L with filtrate of the control (Table 5). From each treatment, samples were collected for chemical determination (50 mL each).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Water flea
- Strain/clone: not indicated
- Source: The test organism, Daphnia magna Straus, originated from the standard laboratory culture cultivated at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology
- Age of parental stock (mean and range, SD): not indicated
- Feeding during test: no

- Acclimation period: no required
- Acclimation conditions (same as test or not): yes
- Type and amount of food: The Daphnia magna were fed with a suspension of algae; mixture of two species Pseudokirchneriella subcapitata: Desmodesmus subspicatus (in 2:1 ratio) originating from separate cultures in the Laboratory of Aquatic Toxicology. Group B vitamins and micronutrients necessary for proper growth were supplied with the lyophilized suspension of Spirulina sp..
- Feeding frequency: according to test guideline
- Health during acclimation (any mortality observed): not indicated

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h

Test conditions

154.1 mg/L as CaCO3
Test temperature:
19.8 - 20.8 °C
7.46 - 7.71
Dissolved oxygen:
7.6 - 8.2
449 µs/cm
Nominal and measured concentrations:
Nominal: 0 (control), filtrate of the test item loading rate of 50 mg/L and 2-fold, 4-fold, 8-fold and 16-fold diluted
measured concentrations at test start: below LoQ (<0.6 mg/L; control) 15.47, 8.04, 4.09, 2.04, 1.01 mg/L
meausred concentrations at test end: below LoQ (<0.6 mg/L; control) 16.51, 8.02, 4.07, 2.04, 1.02 mg/L
At exposure termination determined concentrations of the test item were in the range of 99.44 – 106.7% of the initial concentration, thus confirming stability of Isobutylacetophenon during 48 h of exposure. Therefore, all biological results were referred to the mean measured test concentrations.
Details on test conditions:
- Test vessel: 150 ml glass bottle
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 150 ml
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- Source/preparation of dilution water: M7 medium, according to OECD 202
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily

- Adjustment of pH: no
- Photoperiod: 16.8 h day night regime
- Light intensity: not indicated

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility after 24 and 48 h


Reference substance (positive control):
potassium dichromate

Results and discussion

Effect concentrations
Key result
48 h
Dose descriptor:
Effect conc.:
4.92 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
Results with reference substance (positive control):
48 h EC50: 0.61 mg/L
Reported statistics and error estimates:
The ECx values were calculated with a probit method. The lowest observed effect concentration (LOEC)
and the no observed effect concentration (NOEC) were determined by statistical analyses using the ToxRat Professional commercial software

Applicant's summary and conclusion