Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-07-19 to 1993-08-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Followed guidelines, GLP, analytical.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): C12/14 Alkyl Dimethyl Ethanol Ammonium Chloride (KDB Base) (E-4408.01)
- Molecular formula (if other than submission substance): Not available. Structure provided.
- Molecular weight (if other than submission substance): 307
- Substance type: Pure active substance
- Physical state: Liquid
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Stable
- Storage condition of test material: Stored in dark at room temperature
- Other: None
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples from the control and all test solutions were taken at 0 hr (newly prepared solutions), and at 24 hr (all spent solutions). Samples of ca. 100 ml were taken in glass bottles.
- Sampling method: Aliquots of 3ml formaldehyde (37% v/v) and 0.1ml of a nonionic stock were added to each glass bottle before 100ml of each sample was added. The analysis of these tests solutions was arranged by Procter & Gamble ETC (European Technical Center). At each replacement time about one liter of used control river water was sampled in glass bottles.
- Sample storage conditions before analysis: The bottles were stored in a refrigerator until transfer to Procter & Gamble ETC.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 250mg of the test substance was accurately weighed out and dissolved in one liter of dilution water, giving a solution of 100mg active ingredient per liter (=mg a.i/L). From this solution 15, 27, 48, 84 and 150ml were taken and diluted with dilution water to 1.5 liter, resulting in test solutions of 1.0, 1.8, 3.2, 5.6 and 10.0mg a.i./L. A new concentrated solution was prepared daily throughout the test period.
- Eluate: Not applicable
- Differential loading: Not applicable
- Controls: Filtered river water and DSWL were used as a control media.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Strain: Not available
- Source: The commercial hatchery M.B. Ruysbrcek B.V. (Noordvliet 159, Maassluis)
- Age at study initiation (mean and range, SD): Not available
- Length at study initiation (length definition, mean, range and SD): Average total length 2.6+/-0.2cm
- Weight at study initiation (mean and range, SD): Average total weight 0.13+/-0.02g
- Method of breeding: Not available
- Feeding during test: Not fed
- Food type: Not applicable
- Amount: Not applicable
- Frequency: Not applicable


ACCLIMATION
- Acclimation period: Not available
- Acclimation conditions (same as test or not): Not available
- Type and amount of food: Not available
- Feeding frequency: Not available
- Health during acclimation (any mortality observed): Not available


QUARANTINE (wild caught)
- Duration: Not applicable
- Health/mortality: Not applicable

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
Hardness of the river water was 178 mg/L as CaCO3 (calculated from the level of calcium in the river water, 71 mg/L). Total hardness of the synthetic medium (DSWL) was 224mg/L as CaCO3.
Test temperature:
Temperature in the control media (clean and spent) was measured at 0, 24, 48, 72 and 96h . Temperature ranged 24.7-25.5 (C) throughout the test:
For new control medium (clean): range 24.7-25.2
For control medium just before replacement (spent): 25.0- 25.5
pH:
pH was measured in control medium or test solution just after preparing (clean) and control medium or test solution just before replacement (spent) at 0, 6, 24, 48, 72 and 96h of exposure. Overall, pH ranged 7.8-8.6 throughout the test:
For river water control: range 8.5-8.6(clean), 8.2-8.3(spent)
For DSWL control: range range 8.3-8.4(clean), 7.8-8.1(spent)
For 1.0mg/L: range 8.5-8.6(clean), 8.0-8.3(spent)
For 1.8mg/L: range 8.5-8.6(clean), 8.1-8.3(spent)
For 3.2mg/L: range 8.5-8.6(clean), 8.2(spent)
For 5.6mg/L: range 8.6(clean), 8.2(spent)
For 10.0mg/L: range 8.6(clean), 8.5(spent)
Dissolved oxygen:
DO was measured in control medium or test solution just after preparing (clean) and control medium or test solution just before replacement (spent) at 0, 6, 24, 48, 72 and 96h of exposure. Overall, DO ranged 6.0-8.5mg/L throughout the test (always >60% saturation).
For river water control: range 8.4-8.5mg/L(clean), 7.2-7.6mg/L(spent)
For DSWL control: range range 8.2-8.6mg/L(clean), 6.0-7.9mg/L(spent)
For 1.0mg/L: range 8.3-8.6mg/L(clean), 6.1-8.0mg/L(spent)
For 1.8mg/L: range 8.3-8.6mg/L(clean), 7.0-7.8mg/L(spent)
For 3.2mg/L: range 8.3-8.5mg/L(clean), 7-7.3mg/L(spent)
For 5.6mg/L: range 8.5mg/L(clean), 7.4mg/L(spent)
For 10.0mg/L: range 8.5mg/L(clean), 8.5mg/L(spent)
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal laboratory exposure concentrations based on active ingredient were 0(river water control), 0(DSWL control),1.0, 1.8, 3.2, 5.6 and 10.0 mg/L
Measured concentrations based on active ingredient were 82-107% of nominal (see Table of analytical results below).
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed: open
- Material, size, headspace, fill volume: 2L glass beakers containing 1.5L of control medium or test solution
- Aeration: Aerated
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Daily renewal
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: 10 fish/1.5L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water used was filtered river water. River water was obtained from the river Meuse (Meetstation Keizersveer, Verlengde Napoleonweg 23, Hank). The water was filtered through a set of Sartorius glass fiber prefilters and cellulose nitrate micropore filters with pore size of 3, 1.2, 0.45 and 0.2um. Before and after the filteration the water was stored at room temperature in the dark. The water was caracterized for a number of properties by BCO Analytical Services B.V. Breda, The Netherland under Sterlab conditions. The DSWL water was prepared by addition of several salts to ground water from a locality near Linschoten (the Netherland). The nominal composition contained Na+: 0.33mmol/L, K+: 0.20mmol/L, Ca2+: 1.42mmol/L, Mg2+: 0.82mmol/L, Cl-: 2.81mmol/L, SO4(2-): 0.55mmol/L.
- Total organic carbon: 4.7 mg/L (river water); 1.9 mg/L (DSWL)
- Particulate matter: Not Available
- Metals: See study report for details on metals measured in the river water.
- Pesticides: See study report for details on pesticides measured in the river water.
- Chlorine: Not Available
- Alkalinity: Not Available
- Ca/mg ratio: calcium (71 mg/L) / magnesium (9 mg/L) in the river water; Ca(2)+ : 1.42mmol/L/ Mg(2)+ : 0.82mmol/L in the DSWL.
- Conductivity: Not available
- Culture medium different from test medium: Yes. Fish were cultured in DSWL water, tested in river water.
- Intervals of water quality measurement: Temperature, DO, pH and measured daily in control and each test concentration.



OTHER TEST CONDITIONS
- Adjustment of pH: Not applicable
- Photoperiod: 16h light-8h dark with transition period of ca. 30min in a temperature control room.
- Light intensity: Not available


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality was determined at 0, 6, 24, 48, 72 and 96 h .


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: No
- Test concentrations: Not available
- Results used to determine the conditions for the definitive study: Not available
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
6 h
Dose descriptor:
LC50
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95%CL: 4.3-5.9mg/L
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
4.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95%CL: 3.2-5.6mg/L
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
4.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95%CL: 3.2-5.6mg/L
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
4.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95%CL: 3.2-5.6mg/L
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
4.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95%CL: 3.2-5.6mg/L
Details on results:
- Behavioural abnormalities: For nominal concentration of 5.6mg/L at 6h 3 slow swimming with disturbed equilibrium fishes were observed.
- Observations on body length and weight: None
- Other biological observations: None
- Mortality of control: None
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The LC50 values were calculated assuming a log logistic dose-effect relation. Likehood ratio confidence intervals were derived from the from the confidence intervals.

Any other information on results incl. tables

Mortality in Acute Fish Test of KDB (study # 35662)

Nominal Test Concentration (mg/L)

Fish Mortality (10 per test concentration)

Time 0

24 hrs

48 hrs

72 hrs

96 hrs

0

0

0

0

0

0

1.0

0

0

0

0

0

1.8

0

0

0

0

0

3.2

0

0

0

0

0

5.6

0

10

10

10

10

10.0

0

10

10

10

10

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Criteria of OECD 203 were met: mortality in the control was <10%; DO was >60% of saturation; constant conditions were maintained throughout the test; measured concentrations were 80% of nominal.
Conclusions:
The 96 h acute toxicity of C12/14 Alkyl Dimethyl Ethanol Ammonium Chloride (CAS#85736-63-6) to Brachydanio rerio was 4.2 mg a.i./L, based on nominal concentration of the active ingredient. Measured concentrations were >80% of nominal.
Executive summary:

In a 96 h acute toxicity study, Brachydanio rerio were exposed to C12/14 Alkyl Dimethyl Ethanol Ammonium Chloride (KDB Base) (E-4408.01) at nominal concentrations of 0 (River water control), 0(DSWL water) 1.0, 1.8, 3.2, 5.6 and 10.0 mg a.i./L under semi-static conditions. Mean measured concentrations were 82 -107% of nominal. The test medium was filtered river water (DO >60% saturation throughout the test).

The 96 h LC50 value based on nominal test concentration was 4.2 mg a.i./L with 95% CL of 3.2 to 5.6 mg/L. Measured test concentrations were >80% of nominal.

This toxicity study is classified as acceptable and satisfies the guideline requirement for the OECD 203 fish acute toxicity study.