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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:
12- 14 February 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
No CoA, hence no identity and purity of test substance, potential loss of test substance due to volatilisation, analytical confirmation has been performed but have not been reported when describing results, so results are concluded upon nominal concentrations, which is an underestimation. Also, the use of DMF is not reflecting environmental conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
NAME:
Methyl nonyl ketone (MNK Technical)

SOURCE OF TEST MATERIAL
- Supplied by: Pet and Garden Manufacturing plc

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Required storage condition of test material: Well ventilated 10 - 20 °C
- Actual storage conditions: Room temperature
- Stability under test conditions: Not stated
- Solubility and stability of the test substance in the solvent/vehicle: Practically insoluble (in water)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
10 mg/l stock solution of MNK was prepared by initially dissolving the substance in dimethyl formamide (DMF) prior to dilution in dechlorinated water. The test concentrations were prepared by addition of the appropriate volume of this stock solution to the dilution water to give the required concentrations for the test.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Definitive test concentrations were prepared as 0 (control), 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0mg/l. A further control solution was prepared containing dimethyl formamide at a concentration of 1 ml DMF/l.
- Sampling method: Samples of the test solutions in which the organisms were exposed were taken for analytical verification at the start of the definitive study, before and after renewal of solutions at 24 hours and at the end of the 48 hour exposure period.
- Sample storage conditions before analysis: Test substance specific analysis of each test concentration was carried out as soon as possible after sampling.

Test solutions

Vehicle:
yes
Remarks:
dimethyl formamide
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
MNK is practically insoluble in water, therefore to determine its toxicity to aquatic species a method had to be devised to maintain the sample in a stable aqueous solution. Preliminary experimentation showed that MNK can be dissolved in dimethyl formamide (DMF) to give a clear colourless solution and that when
this was subsequently diluted in algal growth medium a slightly cloudy stable solution could be achieved at concentrations up to 100 mg/l.

For the definitive test, a 10 mg/l stock solution of MNK was prepared by initially dissolving MNK in DMF prior to dilution in the dechlorinated water. The test concentrations were prepared by addition of the appropriate volume of this stock solution to the dilution water to give the required concentrations for the test.

Data available from previous Daphnia flow-through studies had identified the 48 hour EC50 as being approximately 0.54 mg/l and definitive test concentrations were prepared as 0 (control), 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0mg/l. A further control solution was prepared containing dimethyl formamide at a concentration of 1 ml DMF/l.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Test organism: Daphnia magna

Source: Shell Research Laboratories

Date of gravid isolation: 11 February 2002

Age of Daphnia at start of test: Less than 24 hours

Culture conditions:
Temperature: 20 ± 1 °C
Dissolved oxygen: >60% ASV
Photoperiod: 16 hours light and 8 hours dark

Feeding regime: Each working day, a suspension of Chlorella vulgaris at a rate of lmg organic carbon per litre of culture water.

The Daphnia magna for the test were cultured under semi-static conditions and were not fed during the test.

Study design

Test type:
semi-static
Water media type:
freshwater
Remarks:
dechlorinated mains tap water
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
240mg/l CaC03
Test temperature:
21 ± 1°C
pH:
Start: 7.8 - 8.0 after 24hrs 7.7
24hrs new: 7.9-8.0 after 48 hrs 7.7-7.9
Dissolved oxygen:
98 - 100 (% ASV)
Nominal and measured concentrations:
Nominal test concentrations (mg/I): 0; 0.13; 0.25; 0.50; 1.0; 2.0; 4.0
Nominal concentrations were used in the report for the effect assessment, because MNK is a volatile substance of low water solubility and recovery rates did not produce meaningful data. The effect concentration was determined as 2.3 mg/l.
However, arithmetic means were also calculated to conclude more worst-case effect concentrations.
Details on test conditions:
TEST SYSTEM:
-Six test concentrations plus one control and one solvent control (dimethyl formamide) ( 1 mI DMF per litre) each in four replicates (20 animals per test group). 5 Daphnia were placed at random in each dish containing 25 ml of prepared test medium, diluent water only or diluent water plus 1 ml DMF per litre, as appropriate. The dishes were covered with a transparent perspex sheet to limit aerial contamination of the test vessels and reduce evaporative losses.

The test vessels were placed into an incubator at a temperature of 20 ± 1°C with a light cycle of sixteen hours light and eight hours dark and without
supplementary aeration or feeding during the 48 hour exposure period. Temperature, pH values and dissolved oxygen concentration were recorded at the start of the study, before and after renewal of solutions at 24 hours and at the end of the 48-hour exposure period.

EFFECT PARAMETERS MEASURED: The 48 hour EC50 value of MNK Technical to Daphnia magna and the highest no observed effect concentration (NOEC) after 48 hours.
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: concluded in line with evaluation under BPR evaluation
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Basis for effect:
mobility
Remarks on result:
not determinable
Remarks:
There was only 75% immobilisation at the highest test concentration (i.e. 4.0mg/l)
Details on results:
Test substance specific analysis was carried out and it has been shown that MNK disappears from solution over time. The analytical data clearly demonstrated that recovery of MNK from solution varies with typical recovery rates at 0 hours of 47 to 80%. After 24 hours recovery is shown to have dropped significantly to only 0.5 to 7.0%. Similar values were obtained for the replacement solutions at 24 hours, with initial recovery of 38-to 91 % of the nominal value, which dropped to 2 to 5% after the solutions had aged for a further 24 hours.
The water quality measurements of the test solutions were within acceptable limits. Specifically, the dissolved oxygen concentration remained greater than 60% of the air saturation value, which fulfils the validity criteria of the study.

None (0%) of the twenty control Daphnia, maintained in dilution water, or in lml/l DMF were immobilised during the study, and no Daphnia were trapped at the surface of the water in the control vessels, this fulfils the validity criteria of the study which states that control immobilisation must not exceed 10% at the end of the test.
Results with reference substance (positive control):
48h EC50 was 0.9 mg/L (0.8-1.1, 95%CL)

Any other information on results incl. tables

Concentration Nominal (mg/I)     Number immobilised     % immobilisation    
 24 hours  48 hours 24 hours  48 hours 
 0 control  0  0  0  0
DMF control   0  0  0  0
0.13   0  0  0
0.25   0  0  0  0
0.50   0  3  0  15
 1.0  1  6  5  30
2.0  3  6  15  30
4.0   1  15  5  75

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
although the concentration could not be maintained at nominal +/- 20%.
Conclusions:
Based on assessment of geometric mean and arithmetic mean conc, reference is made to the BPR evaluation conclusion, as the decline of substance during the test is significant and expressing the EC on nominal conc would be an underestimation of the actual toxicity: http://dissemination.echa.europa.eu/Biocides/ActiveSubstances/0044-19/Section_7-9.pdf
"The 48-hour EC50 value of Methyl Nonyl Ketone to Daphnia magna was estimated to be 0.23 mg/l (as determined by the Maximum Likelihood-Probit method), within 95% confidence limits of 0.09 mg a.s./l and 8.47 mg a.s./l"

Based on the report and nominal concentrations:
Estimated EC50 value = 2.3 mg/l, (as determined by the Maximum Likelihood-Probit method), 95% confidence limits = 1.6 to 3.9 mg/l
NOEC = 0.50 mg/l (as determined by Fisher's Exact test), LOEC = 1.0 mg/l (as determined by Fisher's Exact test)
Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202. Groups of twenty, Daphnia (less than 24 hours old) were exposed for 48 hours to six concentrations of MNK dispersed in test water with the aid of DMF. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours and the following value was concluded: 48h EC50 0.23 mg/l, based on actual measured (arithmetic mean) concentrations. Measured concentrations ranged from 47 - 80 % of nominal at 0 hours and 2 - 7 % of nominal at 24 hours; renewed 24h solutions showed 38 - 91% of nominal declining to 2 - 5% at 48h .