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Short-term toxicity to aquatic invertebrates

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short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20th January to 19th February 2016
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
The test was conducted according to an appropriate guideline under GLP conditions. It has been assigned reliability 2 due to the non-specific analysis (TOC) of the test substance concentrations.
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
Analytical monitoring:
Details on sampling:
- Concentrations: 10, 18, 32, 56 and 100 mg/L.
- Sampling method: Specific analyses proved not possible and, after consultation with the sponsor, it was decided to analyse the Total Organic Carbon (TOC) concentration as a surrogate measure.
Singular samples for TOC-analysis were taken from all test concentrations without algae and the control without algae according to the schedule below:
Frequency at t=0 h, t=24 h and t=72 h
Volume 40 mL
Additionally, singular reserve samples of 40 mL were taken from all test solutions without algae for possible analysis. If not already used, these samples were stored in a refrigerator for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
- Sample storage conditions before analysis: Storage Samples were stored in a refrigerator until analysis.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The batch of CF-73 tested was a clear light yellow liquid and completely soluble in test medium at the tested concentrations. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with the highest test concentration, i.e. 100 mg/L. No special treatment other than careful mixing was required to dissolve the test item in medium. After adjusting the pH of the resulting solution from 9.5 to 8.7 (combined limit/range-finding test) or from 9.7 to 8.8 (final test) with 1N HCl (Merck, Darmstadt, Germany), lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All final test solutions were clear and colourless.
- Controls: Test medium without test item or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All final test solutions were clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna
- Strain: Straus, 1820
- Source: In-house laboratory culture with a known history
- Age at study initiation (mean and range, SD): <24 hours
- Method of breeding: pathogenesis
- Feeding during test - none

- Health during acclimation (any mortality observed): Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Test temperature:
Start: 8.0 - 8.7
48 h: 8.1 - 8.3
Dissolved oxygen:
9.2 - 9.7
Nominal and measured concentrations:
Nominal: Control, 10, 18, 32, 56 and 100 mg/L.
Measured: see table 1 in Results
Details on test conditions:
- Test vessel: 100 mL, all-glass
- Type (delete if not applicable): open / closed: not reported
- Material, size, headspace, fill volume: all-glass, 100 ml, 20 ml, 80 ml
- Aeration: No
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: not reported

- Source/preparation of dilution water: M7 medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen and pH: At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.

- Adjustment of pH: yes
- Photoperiod: 16 hours photoperiod daily
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility (including mortality): At 24 hours and at 48 hours.

- Spacing factor for test concentrations: approximately 1.8
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: immobility - no immobility at concentrations up to and including 10 mg/l. 90% Immobility observed at 100 mg/l.
Reference substance (positive control):
potassium dichromate
Key result
48 h
Dose descriptor:
Effect conc.:
51 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95%-confidence interval: 44-59 mg/l
Details on results:
- Behavioural abnormalities: none reported
- Observations on body length and weight: none reported
- Other biological observations: none reported
- Mortality of control: none
- Other adverse effects control: none reported
- Abnormal responses: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Mortality: Yes
- EC50/LC50: The 48h-EC50 was 0.38 mg/L with a 95% confidence interval between 0.33 and 0.43 mg/L.
- Other:

Table 1: Measured concentrations versus nominal concentrations during the final test

 Nominal concentration CF-73 (mg/L)    Measured concentration (TOC, mg/L)      % relative to nominal at t = 0 h   
 t = 0 h  t = 48 h
 Control  0.6241  0.5981  n.a
 10 (5.6)  5.84 6.03   104
 18 (10)  10.5 10.3  104
 32 (18)  18.1  18.1  101
 56 (31)  31.3  31.0  100
 100 (56)  56.5  55.9  101

( ): nominal TOC concentration in mg C/L

n.a.: not applicable

1: Values below 1 mg C/L should be considered indicative

Table 2: Number of introduced daphnids and incidence of immobility in the final test

 Time (h)          Nominal concentration CF-73 (mg/L)               
 Control  10  18  32  56  100
 0  Total no. Daphnia Introduced  20  20  20  20  20  20
 24     Total immobilised  0  0  0  2  5
 % effect  0  0  0  0  10  25
 48     Total immobilised  1  0  0  1  14  19
 % effect  5  0  0  5  70  95
Validity criteria fulfilled:
A 48 hour EC50 value of 51 mg/l has been determined for the effects of the submission substance on mobility of Daphnia magna, based on analytically (TOC) confirmed nominal concentrations.

Description of key information

Toxicity to aquatic invertebrates: 48 hour EC50: 51 mg/L (nominal), for the effects of the test substance on immobilisation of Daphnia magna, in accordance with guideline OECD 202. The rapid hydrolysis rates of the constituents of the test substance means that the Daphnia were predominantly exposed to the hydrolysis products of the test substance.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
51 mg/L

Additional information

A 48 hour EC50 of 51 mg/L (analytically confirmed (TOC) nominal concentration) has been determined for the effects of the submission substance on immobilisation of Daphnia magna. Each constituent of the substance hydrolyses rapidly or very rapidly in contact with water, therefore it is likely that the Daphnia were exposed to the hydrolysis products of the test substance.

Verification of test concentrations was carried out by Total Organic Carbon (TOC) analysis.

The TOC content of the test substance was estimated to be 56% based on the test item information provided by the Sponsor.

Samples taken from nominally 100 mg/L were analysed. The initial TOC concentration was 54 mg/L, corresponding to a test item concentration of approximately 96 mg/L, which remained stable during the test period (98% of initial after 48 hours).