Registration Dossier

Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-06-28 to 1994-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, followed Guideline, well documented, with analytical.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
yes
Remarks:
very minor- no transition between dusk and dawn
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)
- Physical state: Colorless liquid
- Chainlength distribution: C12 (69.8%), C14 (25.4%), C16 (4.8%).
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
N/A

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations
- Sampling method: twice per week, (3, 8, 15, 22, and 28 days) approx 1 L taken from all solutions and transferred to glass bottles, preserved w/ 30ml formaldehyde and 1ml 0.5% nonionic stock solution.
- Sample storage conditions before analysis: Refrigerated until transfer to Sponsor. Sponsor responsible for chemical analyses.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct weighing of test substance into flask, diluted with appropriate amount of dilution water. Stock solutions were prepared in one batch for the whole test and stored in the refrigerator.
- Eluate:N/A
- Differential loading: N/A
- Controls: DSWL
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc): N/A

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Brachydanio rerio
- Strain: N/A
- Source: Commercially obtained
- Pre-exposure reproductive performance : N/A


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): In six basins 1 female was kept with 3 males separated by mesh.
- Method of collection of fertilised eggs: As soon as eggs were deposited they were collected an put into test vessels.
- Subsequent handling of eggs: Stage of embryonic development at the start of test was verified under a microscope as blastula stage.


POST-HATCH FEEDING
- Start date: Immediately
- Type/source of feed: Rotifers, then starting day 10, Artemia nauplii enriched with Selco
- Amount given: abundantly
- Frequency of feeding: daily

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Post exposure observation period:
None

Test conditions

Hardness:
185 mg/L CaCO3
Test temperature:
25.0-25.5 C
pH:
7.8-8.3 SU
Dissolved oxygen:
7.0-8.6 mg/L
Salinity:
N/A
Nominal and measured concentrations:
Nominal- Control (0), 0.056, 0.10, 0.18, 0.32, 0.56, 1.0, and 1.8 mg active ingredient/L.
Measured-See Tables inserted into "Any other information on materials and methods incl. tables" below. Overall, the measured concentrations were 24% of nominal.
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): N/A
- Test vessel: N/A
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, approx. 226 ml volume, with nylon screens on the bottom
- Aeration: no
- Type of flow-through: Van Leeuwen diluter
- Renewal rate of test solution (frequency/flow rate): 250 mls per 60 min cycle flow
- No. of fertilized eggs per vessel:50 at test initiation, reduced to 20 at 24 hr
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: DSWL, prepared from groundwater
- Total organic carbon: N/A
- Particulate matter: N/A
- Metals: N/A
- Pesticides: N/A
- Chlorine: N/A
- Alkalinity: N/A
- Ca/mg ratio: N/A
- Conductivity: N/A
- Salinity: N/A
- Culture medium different from test medium: No
- Intervals of water quality measurement: 24 hr


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16hr light/ 8 hr dark
- Light intensity: N/A


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and growth 24 hr


VEHICLE CONTROL PERFORMED: no


RANGE-FINDING STUDY
- Test concentrations: N/A
- Results used to determine the conditions for the definitive study: N/A
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
0.23 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.66 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
number hatched
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.66 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
length
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.66 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
weight
Details on results:
- Mortality/survival at embryo and larval stages:
- Overall mortality/survival: 19 of 80 fish survived at 28 days in the highest concentration; 5% mortality in the control at the end of the test. See Table 1 below.
- Days to hatch and numbers hatched: all eggs in all concentrations hatched within 6 days
- Data for length and weight of surviving fish: See Table 1 below.
- Type of and number with morphological abnormalities: Nothing beyond normal except in the highest concentration.
- Type of and number with behavioural abnormalities: N/A
- Other biological observations: N/A
- Effect concentrations exceeding solubility of substance in test medium: None
- Incidents in the course of the test which might have influenced the results: None
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
Statistical significance for mortality was determined with a binomial test at the 95% anfd 99% significance level, combining the results of the replicates.Statistical significance for growth was determined with the two-tailed Dunnett's test at 95% and 99%. Observations at each concentration were compared with the control. LC50 values and their confidence intervals were calculated by means of a parametric model developed by Kooijman.

Any other information on results incl. tables

Table 1. Summary of results on hatching, growth, and mortality of eggs/larvae of Brachydanio rerio exposed to KDB base.

Nominal Concentration (mg a.i./L)

% eggs hatched after 5 d

% mortality after 28 d

No. of  fish

Length (cm)

Wet Weight (mg)

Dry Weight (mg)

0

99

5.0

76

1.03±0.17

7.96±3.25

1.53±0.12

0.056

100

16.3

67

1.08±0.13

8.77±3.24

1.73±0.26

.10

99

7.5

74

1.05±0.15

8.26±3.48

1.52±0.18

0.18

100

8.8

73

1.04±0.15

8.14±3.08

1.57±0.054

0.32

100

12.5

70

1.05±0.12

8.16±2.67

1.55±0.099

0.56

100

10

72

1.03±0.14

7.60±3.03

1.48±0.29

1.0

98

22.5*

62

1.07±0.12

8.95±3.55

1.75±0.23

1.8

95

76.3*

19

1.01±0.30

8.99±8.48

2.01±0.96

* Significantly different from the control.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Guideline criteria fulfilled
Conclusions:
The 28 day NOEC of the test substance (KDB) to Brachydanio rerio was 0.16 mg a.i./L, based on measured test concentration.
Executive summary:

The chronic effect of the test substance (C12 -14 dimethyl hydroxyethyl ammonium chloride) (KDB-base) on Brachydanio rerio was evaluated in an early life-stage chronic toxicity test. The test was conducted according to OECD draft guideline 210, and GLP principles.

The nominal test concentrations were 0 (control), 0.056, 0.10, 0.18, 0.32, 0.56, 1.0, and 1.8 mg active ingredient/L. Four replicates per concentration were used, the exposure duration was 28 days, and the test used an intermittent flow-through system. Endpoints included hatching, mortality, growth, and egg/larvae malformations.

The mean measured test substance concentrations were 0.027 (control), 0.027, 0.029, 0.032, 0.042, 0.109, 0.156, and 0.439 mg/L. Corrected for the % recovery of the analytical method, the 3 highest test concentrations were 0.16, 0.23, and 0.66 mg/L. Overall, the measured concentrations were 24% of nominal. Therefore, the test results are reported on the basis of measured concentration.

Hatching of the eggs was not affected at any of the test concentrations. Malformations were observed only at the highest test concentration. Mortality was significantly increased at 0.23 mg/L (measured). Growth was not affected at any test concentration.

The NOEC = 0.16 mg/L (measured concentration), based on the lowest NOEC for the criteria quantitatively (hatching, mortality and growth) and qualitatively (condition) determined in this study. The LOEC was 0.23 mg/L.

This toxicity test is classified as acceptable and satisfies the guideline requirements for the OECD 210 fish early life stage chronic toxicity test.